Tmem100 peptides and variants thereof and their use in treating or preventing diseases or conditions

ABSTRACT

The present invention features compositions of Tmem100 peptides and variants thereof, and their use in treating or preventing diseases or conditions.

RELATED APPLICATIONS

This application claims priority to U.S. Ser. No. 61/943,615, filed onFeb. 24, 2014, which is incorporated herein by reference in itsentirety.

STATEMENT OF RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH

This work was supported by grant number 1R01GM087369, awarded by theNational Institutes of Health. The Government has certain rights in theinvention.

BACKGROUND OF THE INVENTION

Hypersensitivity to thermal, mechanical, and chemical stimuli is one ofthe main symptoms of many debilitating diseases, including a variety ofpainful conditions (e.g., arthritis, tissue damage, post-operativestate, etc.), pulmonary conditions (e.g., asthma and COPD), and lowerurinary tract disorders. Neuronal plasticity within peripheral sensoryganglia plays a key role in hypersensitivity. It is recognized that awide range of external stimuli causing hypersensitivity are detected bysensory ganglia neurons. This diverse range of stimuli reflects theheterogeneous nature of a similarly diverse set of molecules needed todistinguish them, and Transient Receptor Potential (TRP) channels playan important role in such sensory modalities. Given the centralimportance of ion channels in modulating membrane potential and ion fluxin cells, identification of agents that can promote or inhibitparticular ion channels are of great interest as research tools and aspossible therapeutic agents.

One such channel is the Transient Receptor Potential A1 (TRPA1) channel.TRPA1 is a calcium permeable channel, specifically a non-selectivecalcium permeable cation channel. In addition to calcium ions, TRPA1channels are permeable to other cations, for example sodium. Thus. TRPA1channels modulate membrane potential by modulating the flux of cationssuch as calcium and sodium ions. TRPA1 is found in sensory neurons andfunctions as a signal transduction receptor linking inflammation topain. Activation of TRPA1 can cause pain by inducing firing ofnociceptive neurons and driving central sensitization in the spinalcord. TRPA1 stimulation can also increase firing of sensory neurons,leading to the release of pro-inflammatory neuropeptides such as NK-A,substance P and CGRP (which induce vasodilation and help recruit immunecells). A variety of endogenous reactive compounds produced duringinflammation activate TRPA1 (including 4-hydroxynonenal released duringliposome peroxidation; cyclopentane prostaglandins synthesized by COXenzymes; hydrogen peroxide produced by oxidative stress). TRPA1 can alsobe activated by a variety of stimuli, including natural products,environmental irritants, amphipathic molecules and pharmacologicalagents. Activation of TRPA1 also sensitizes TRPA1 to cold. Furthermore,a gain-of-function mutation in TRPA1 causes familial episodic painsyndrome; patients suffering from this condition have episodic pain thatare triggered by cold. Thus, TRPA1 is believed to play a role in pain,including pain related to nerve damage, cold allodynia and inflammatorypain.

Since the mis-regulation of ion channels is often associated withpathological conditions, it would be desirable to identify and makepeptides or fragments thereof that can modulate one or more functions ofion channels including TRP channels. Such peptides and fragments thereofhave a variety of in vitro and in vivo uses for treating and preventingconditions, such as pain and itch.

SUMMARY OF THE INVENTION

TRPA1, an ion channel expressed in pain sensing neurons, plays anessential role in pain and itch. The present invention is based upon thefinding that Tmem100 (also known as Pirt2 and used interchangeablyherein), a membrane protein, functions as a regulator of TRPA1. Thepresent invention discloses a cell permeable peptide (CPP) derived fromTmem100 mutant protein (T100-Mut; also known as P2-MutCPP and usedinterchangeably herein) that strongly inhibits the activity of TRPA1 andblocks acute and chronic pain. Accordingly, the present inventiondescribes therapeutic agents for pain and itch.

The invention features a method for treating or preventing a conditionassociated with TRPA1 function or for which reduced TRPA1 activity canreduce the severity, comprising administering an effective amount of aTmem100 mutant polypeptide, or fragment thereof. For example, theeffective amount of the Tmem100 mutant polypeptide comprises a 0.1-1,000μl dose of 0.1-1,000 μM human T100-Mut peptide, e.g., 10 μl dose of10-200 μM human T100-Mut peptide. Ultimately, the attending physician orveterinarian will decide the appropriate amount and dosage regimen. TheTmem100 mutant polypeptide is administered once per month, twice permonth, once per week, twice per week, once per day or every 12 hours,every 8 hours, every 4 hours, every 2 hours, or every hour.

The invention features a method of preventing, treating, or alleviatingsymptoms of a disease or condition associated with TRPA1 function or forwhich reduced TRPA1 activity can reduce the severity, comprisingadministering to a subject in need thereof a Tmem100 mutant polypeptide,or fragment thereof.

The invention features a method of inhibiting TRPA1 function in a cell,comprising administering to the cell an effective amount of a Tmem100mutant polypeptide, or fragment thereof, thereby inhibiting TRPA1function in the cell.

The TRPA1 function is an association with TRPV1. The Tmem100 mutantpolypeptide, or fragment thereof, enhances the association of TRPA1 withTRPV1.

The TRPA1 function is an inward TRPA1-mediated current, an outwardTRPA1-mediated current, TRPA1-mediated ion flux or TRPA1-mediatedneuronal hyperexcitability.

The Tmem100 mutant polypeptide comprises a polypeptide with one or morealterations in the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2,or fragments thereof.

The Tmem100 mutant polypeptide comprises an amino acid sequence selectedfrom the group consisting of: SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7,SEQ ID NO: 8, SEQ ID NO: 17, or a fragment thereof.

The Tmem100 mutant polypeptide, or fragment thereof, is provided to acell and the cell is a sensory neuron.

The cell body of the sensory neuron resides in the dorsal root ganglia(DRG).

The method is used to prevent, treat, or alleviate symptoms of pain.

The method is used to prevent, treat, or alleviate symptoms of itch. Inone embodiment, the pain is acute pain or chronic pain.

The Tmem100 mutant polypeptide, or fragment thereof, is administered incombination with one or more agents.

The Tmem100 mutant polypeptide, or fragment thereof, is administered incombination with one or more of a TRPV1 inhibitor, a TRPV3 inhibitor, aTRPV4 inhibitor, or a TRPM8 inhibitor.

The invention features a pharmaceutical agents for treating orpreventing a condition involving activation of TRPA1 or for whichreduced TRPA1 activity can reduce the severity, comprising an effectiveamount of a Tmem100 mutant polypeptide, or fragment thereof.

The Tmem100 mutant polypeptide comprises a polypeptide with one or morealterations in the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2,or fragments thereof.

The Tmem100 mutant polypeptide comprises an amino acid sequence selectedfrom the group consisting of: SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7and SEQ ID NO: 8, or fragments thereof.

In another aspect, the invention features an isolated polypeptidecomprising an amino acid sequence selected from the group consisting of:SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8, or fragmentsthereof.

The invention features an isolated polypeptide encoded by a nucleic acidsequence comprising a nucleotide sequence selected from the groupconsisting of: SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO:12, or fragments thereof.

The invention features an isolated nucleic acid comprising a nucleotidesequence selected from the group consisting of: SEQ ID NO: 9, SEQ ID NO:10, SEQ ID NO: 11 and SEQ ID NO: 12, or fragments thereof.

The invention features an isolated nucleic acid comprising a nucleotidesequence which encodes a polypeptide comprising an amino acid sequenceselected from the group consisting of: SEQ ID NO: 5, SEQ ID NO: 6, SEQID NO: 7 and SEQ ID NO: 8, or fragments thereof.

The invention features an expression vector, which replicates in atleast one of a prokaryotic cell and eukaryotic cell, comprising thenucleic acid of any one of the above aspects.

The invention features a cell comprising the expression vector of claimand expressing said polypeptide.

The invention features a method of producing a polypeptide comprisingculturing the cell of in a cell culture medium to express saidpolypeptide.

Definitions

As used herein, the singular forms “a”, “an”, and “the” include pluralforms unless the context clearly dictates otherwise.

Unless specifically stated or obvious from context, as used herein, theterm “or” is understood to be inclusive.

As used herein, the terms “comprises,” “comprising,” “containing,”“having” and the like can have the meaning ascribed to them in U.S.Patent law and can mean “includes,” “including,” and the like;“consisting essentially of” or “consists essentially” likewise has themeaning ascribed in U.S. Patent law and the term is open-ended, allowingfor the presence of more than that which is recited so long as basic orcharacteristics of that which is recited is not changed by the presenceof more than that which is recited, but excludes prior art embodiments.

As used herein, the term “administering” is defined herein as a means ofproviding an agent or a composition containing the agent to a subject ina manner that results in the agent being inside the subject's body. Suchan administration can be by any route including, without limitation,oral, transdermal (e.g., vagina, rectum, oral mucosa), by injection(e.g., subcutaneous, intravenous, parenterally, intraperitoneally,intrathecal, intradermal), by topical application, or by inhalation(e.g., oral or nasal). Pharmaceutical preparations are, of course, givenby forms suitable for each administration route.

As used herein, the term “agent” is meant small molecule chemicalcompound, antibody, nucleic acid molecule, or polypeptide, or fragmentsthereof. The terms “compound” and “agent” are used interchangeably torefer to the peptides or fragments thereof of the invention. Thecompounds of the present invention are peptides or fragments thereof.Such compounds can bind to and inhibit a function of TRPA1. Otherexemplary compounds are nucleic acids, for example, TRPA1 antisenseoligonucleotides or TRPA1 RNAi constructs. Such compounds can inhibitthe expression of TRPA1, thereby inhibiting the activity of TRPA1. Otherexemplary compounds that act as inhibitors include ribozyme. As usedherein, the term “composition” is meant any compound or agent of theinvention in pharmaceutical formulation and is used interchangeably withcompound and agent.

As used herein, the term “analog” is meant an agent that is notidentical, but has analogous functional or structural features. Anexemplary analog is a peptide, peptide fragment or mutant with similarbioactive, physical, or chemical properties to agents of the invention.

As used herein, the terms “antagonist” and “inhibitor” are usedinterchangeably to refer to an agent that decreases or suppresses abiological activity, such as to repress an activity of an ion channel,such as TRPA1. TRPA1 inhibitors include inhibitors having anycombination of the structural and/or functional properties disclosedherein.

As used herein, the term “combination” embraces groups of compounds ornon-drug therapies useful as part of a combination therapy.

As used herein, the term “disease” is meant any condition or disorderthat damages or interferes with the normal function of a cell, tissue,or organ.

As used herein, an “effective amount” of, e.g., a TRPA1 antagonist, withrespect to the subject methods of inhibition or treatment, refers to anamount of the antagonist in a preparation which, when applied as part ofa desired dosage regimen brings about a desired clinical or functionalresult. Without being bound by theory, an effective amount of a TRPA 1antagonist of the present invention, includes an amount of a TRPA1antagonist effective to decrease one or more in vitro or in vivofunction of a TRPA1 channel. Exemplary functions include, but are notlimited to, membrane polarization (e.g., an antagonist promoteshyperpolarization of a cell), ion flux, ion concentration in a cell,outward current, and inward current. Agents that antagonize TRPA1function include agents that antagonize an in vitro or in vivofunctional activity of TRPA1. When a particular functional activity isonly readily observable in an in vitro assay, the ability of an agent toinhibit TRPA1 function in that in vitro assay serves as a reasonableproxy for the activity of that agent. An effective amount is an amountsufficient to inhibit a TRPA1-mediated current and/or the amountsufficient to inhibit TRPA1 mediated ion flux.

The TRPA1 inhibitors of the present invention are characterizedaccording to their activity, or lack of activity, against one or moreother ion channels. When other ion channels are referred to, inhibitionof a function of such other ion channels is defined similarly. Forexample, inhibition of an ion channel or an activity of an ion channelmeans the antagonist inhibits one or more functional activities of theother ion channel. Such functions include the current mediated by theparticular ion channel, ion flux, or membrane polarization.

By “fragment” is meant a portion of a polypeptide or nucleic acidmolecule. This portion contains at least 10%, 20%, 30%, 40%, 50%, 60%,70%, 80%, or 90% of the entire length of the reference nucleic acidmolecule or polypeptide. A fragment contains 10, 20, 30, 40, 50, 60, 70,80, 90, or 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000nucleotides or amino acids.

As used herein, the term “nucleic acid” refers to a polymeric form ofnucleotides, either ribonucleotides or deoxynucleotides or a modifiedform of either type of nucleotide. The terms should also be understoodto include, as equivalents, analogs of either RNA or DNA made fromnucleotide analogs, and, as applicable to the embodiment beingdescribed, single-stranded (such as sense or antisense) anddouble-stranded polynucleotides.

As used herein, the term “pharmaceutically acceptable” refers toapproved or approvable by a regulatory agency of the Federal or a stategovernment or listed in the U.S. Pharmacopeia or other generallyrecognized pharmacopeia for use in animals, including humans.

As used herein, the term “preventing” is art-recognized, and when usedin relation to a condition, such as a local recurrence (e.g., pain), adisease such as cancer, a syndrome complex such as heart failure or anyother medical condition, is well understood in the art, and includesadministration of an agent which reduces the frequency of, or delays theonset of, symptoms of a medical condition in a subject relative to asubject which does not receive the agent. Thus, prevention of cancerincludes, for example, reducing the number of detectable cancerousgrowths in a population of patients receiving a prophylactic treatmentrelative to an untreated control population, and/or delaying theappearance of detectable cancerous growths in a treated populationversus an untreated control population, e.g., by a statistically and/orclinically significant amount. Prevention of an infection includes, forexample, reducing the number of diagnoses of the infection in a treatedpopulation versus an untreated control population, and/or delaying theonset of symptoms of the infection in a treated population versus anuntreated control population. Prevention of pain includes, for example,reducing the magnitude of, or alternatively delaying, pain sensationsexperienced by subjects in a treated population versus an untreatedcontrol population.

“Peptides,” “polypeptides,” and “oligopeptides” are chains of aminoacids (typically L-amino acids; however, D-amino acids are alsocontemplated) whose alpha carbons are linked through peptide bondsformed by a condensation reaction between the carboxyl group of thealpha carbon of one amino acid and the amino group of the alpha carbonof another amino acid. In some cases, the terminal amino acid at one endof the chain (for example, the amino terminal) has a free amino group,while the terminal amino acid at the other end of the chain (forexample, the carboxy terminal) has a free carboxyl group. As such, theterm “amino terminus” (abbreviated N-terminus) refers to the freealpha-amino group on the amino acid at the amino terminal end of thepeptide, or to the alpha-amino group (imino group when participating ina peptide bond) of an amino acid at any other location within thepeptide. The term “carboxy terminus” (abbreviated C-terminus) refers tothe free carboxyl group on the amino acid at the carboxy terminal end ofa peptide, or to the carboxyl group of an amino acid at any otherlocation within the peptide.

Typically, the amino acids making up a peptide are numbered in order,starting at the amino terminus and increasing in the direction towardthe carboxy terminus of the peptide. Thus, when one amino acid is saidto “follow” another, that amino acid is positioned closer to the carboxyterminal end of the peptide than the preceding amino acid.

By “polypeptide” is meant a polymer in which the monomers are amino acidresidues that are joined together through amide bonds. When the aminoacids are alpha-amino acids, either the L-optical isomer or theD-optical isomer can be used, the L-isomers being preferred in nature.The term “polypeptide” or “protein” as used herein encompasses any aminoacid sequence and includes, but may not be limited to, modifiedsequences such as glycoproteins or amidated proteins. The term“polypeptide” is specifically intended to cover naturally occurringproteins, as well as those that are recombinantly or syntheticallyproduced. Exemplary polypeptides include gene products,naturally-occurring proteins, homologs, orthologs, paralogs, fragments,and other equivalents, variants and analogs of the foregoing.

In some cases, reference to “peptide” or “polypeptide” when in referenceto any polypeptide of this invention, is meant to include nativepeptides (either degradation products, synthetically synthesizedpeptides or recombinant peptides) and peptidomimetics (typically,synthetically synthesized peptides), such as peptoids and semipeptoidswhich are peptide analogs, which may have, for example, modificationsrendering the peptides more stable while in a body or more capable ofpenetrating into cells. Such modifications include, but are not limitedto N terminal, C terminal, or peptide bond modification, including, butnot limited to, backbone modifications, and residue modification, eachof which represents an additional embodiment of the invention. Methodsfor preparing peptidomimetic compounds are well known in the art and arespecified, for example, in Quantitative Drug Design, C. A. Ramsden Gd.,Chapter 17.2, F. Choplin Pergamon Press (1992).

As used herein, the term “sequence identity” means that sequences areidentical (i.e., on a nucleotide-by-nucleotide basis for nucleic acidsor amino acid-by-amino acid basis for polypeptides) over a window ofcomparison. The term “percentage of sequence identity” is calculated bycomparing two optimally aligned sequences over the comparison window,and multiplying the result by 100 to yield the percentage of sequenceidentity. Methods to calculate sequence identity are known to those ofskill in the art and described in further detail below.

As used herein, the terms “stringent conditions” or “stringenthybridization conditions” refer to conditions which promote specifichybridization between two complementary polynucleotide strands so as toform a duplex. Stringent conditions can be selected to be about 5° C.lower than the thermal melting point (Tm) for a given polynucleotideduplex at a defined ionic strength and pH. The length of thecomplementary polynucleotide strands and their GC content will determinethe Tm of the duplex, and thus the hybridization conditions necessaryfor obtaining a desired specificity of hybridization. The Tm is thetemperature (under defined ionic strength and pH) at which 50% of the apolynucleotide sequence hybridizes to a perfectly matched complementarystrand. In certain cases it is desirable to increase the stringency ofthe hybridization conditions to be about equal to the Tm for aparticular duplex. In certain cases, stringent hybridization conditionsinclude a wash step of 0.2×SSC at 65° C.

The term “patient” or “subject” refers to an animal which is the objectof treatment, observation, or experiment. By way of example only, asubject includes, but is not limited to, a mammal, including, but notlimited to, a human or a non-human mammal, such as a non-human primate,bovine, equine, canine, ovine, or feline.

Polynucleotides, polypeptides, or other agents are purified and/orisolated. Specifically, as used herein, an “isolated” or “purified”nucleic acid molecule, polynucleotide, polypeptide, or protein, issubstantially free of other cellular material, or culture medium whenproduced by recombinant techniques, or chemical precursors or otherchemicals when chemically synthesized. Purified compounds are at least60% by weight (dry weight) the compound of interest. Preferably, thepreparation is at least 75%, more preferably at least 90%, and mostpreferably at least 99%, by weight the compound of interest. Forexample, a purified compound is one that is at least 90%, 91%, 92%, 93%,94%, 95%, 98%, 99%, or 100% (w/w) of the desired compound by weight.Purity is measured by any appropriate standard method, for example, bycolumn chromatography, thin layer chromatography, or high-performanceliquid chromatography (HPLC) analysis. A purified or isolatedpolynucleotide (ribonucleic acid (RNA) or deoxyribonucleic acid (DNA))is free of the genes or sequences that flank it in itsnaturally-occurring state. A purified or isolated polypeptide is free ofthe amino acids or sequences that flank it in its naturally-occurringstate. Purified also defines a degree of sterility that is safe foradministration to a human subject, e.g., lacking infectious or toxicagents.

Similarly, by “substantially pure” is meant a nucleotide or polypeptidethat has been separated from the components that naturally accompany it.Typically, the nucleotides and polypeptides are substantially pure whenthey are at least 60%, 70%, 80%, 90%, 95%, or even 99%, by weight, freefrom the proteins and naturally-occurring organic molecules with theyare naturally associated.

“Conservatively modified variations” of a particular polynucleotidesequence refers to those polynucleotides that encode identical oressentially identical amino acid sequences, or where the polynucleotidedoes not encode an amino acid sequence, to essentially identicalsequences. Because of the degeneracy of the genetic code, a large numberof functionally identical nucleic acids encode any given polypeptide.For instance, the codons CGU, CGC, CGA, CGG, AGA, and AGG all encode theamino acid arginine. Thus, at every position where an arginine isspecified by a codon, the codon can be altered to any of thecorresponding codons described without altering the encoded polypeptide.Such nucleic acid variations are “silent substitutions” or “silentvariations,” which are one species of “conservatively modifiedvariations.” Every polynucleotide sequence described herein whichencodes a polypeptide also describes every possible silent variation,except where otherwise noted. Thus, silent substitutions are an impliedfeature of every nucleic acid sequence which encodes an amino acid. Oneof skill will recognize that each codon in a nucleic acid (except AUG,which is ordinarily the only codon for methionine) can be modified toyield a functionally identical molecule by standard techniques.

Similarly, “conservative amino acid substitutions,” in one or a fewamino acids in an amino acid sequence are substituted with differentamino acids with highly similar properties are also readily identifiedas being highly similar to a particular amino acid sequence, or to aparticular nucleic acid sequence which encodes an amino acid. Suchconservatively substituted variations of any particular sequence are afeature of the present invention. Individual substitutions, deletions oradditions which alter, add or delete a single amino acid or a smallpercentage of amino acids (typically less than 5%, more typically lessthan 1%) in an encoded sequence are “conservatively modified variations”where the alterations result in the substitution of an amino acid with achemically similar amino acid. Conservative substitution tablesproviding functionally similar amino acids are well known in the art.See, e.g., Creighton (1984) Proteins, W.H. Freeman and Company,incorporated herein by reference. The following six groups are examplesof amino acids that are considered to be conservative substitutions forone another: 1) Alanine (A), Serine (S), Threonine (T); 2) Aspartic acid(D), Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine(R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine(V); and 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).

A non-conservative amino acid substitution can result from changes in:(a) the structure of the amino acid backbone in the area of thesubstitution; (b) the charge or hydrophobicity of the amino acid; or (c)the bulk of an amino acid side chain. Substitutions generally expectedto produce the greatest changes in protein properties are those inwhich: (a) a hydrophilic residue is substituted for (or by) ahydrophobic residue; (b) a proline is substituted for (or by) any otherresidue; (c) a residue having a bulky side chain, e.g., phenylalanine,is substituted for (or by) one not having a side chain, e.g., glycine;or (d) a residue having an electropositive side chain, e.g., lysyl,arginyl, or histadyl, is substituted for (or by) an electronegativeresidue, e.g., glutamyl or aspartyl.

As used herein, the twenty conventional amino acids and theirabbreviations follow conventional usage. See Immunology—A Synthesis (2ndEdition, E. S. Golub and D. R. Gren, Eds., Sinauer Associates,Sunderland7 Mass. (1991)). Stereoisomers (e.g., D-amino acids) of thetwenty conventional amino acids, unnatural amino acids such as α-,α-disubstituted amino acids, N-alkyl amino acids, lactic acid, and otherunconventional amino acids may also be suitable components forpolypeptides of the present invention. Examples of unconventional aminoacids include: 4 hydroxyproline, γ-carboxyglutamate,ε-N,N,N-trimethyllysine, ε-N-acetyllysine, O-phosphoserine,N-acetylserine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysine,σ-N-methylarginine, and other similar amino acids and imino acids (e.g.,4-hydroxyproline). In the polypeptide notation used herein, theleft-hand direction is the amino terminal direction and the right-handdirection is the carboxy-terminal direction, in accordance with standardusage and convention.

Peptide analogs are commonly used in the pharmaceutical industry asnon-peptide drugs with properties analogous to those of the templatepeptide. These types of non-peptide compound are termed “peptidemimetics” or “peptidomimetics”. Fauchere, J. Adv. Drug Res. 15:29(1986), Veber and Freidinger TINS p. 392 (1985); and Evans et al. J.Med. Chem. 30:1229 (1987). As used herein “peptide mimetics” refers toorganic compounds which are structurally similar to peptides. Thepeptide mimetics are typically designed from existing peptides to alterthe molecule's characteristics. Improved characteristics can involve,for example, improved stability such as resistance to enzymaticdegradation, or enhanced biological activity, improved affinity byrestricted preferred conformations and ease of synthesis. Structuralmodifications in the peptidomimetic in comparison to a peptide, caninvolve backbone modifications as well as side chain modification. Suchcompounds are often developed with the aid of computerized molecularmodeling.

Peptide mimetics that are structurally similar to therapeutically usefulpeptides may be used to produce an equivalent therapeutic orprophylactic effect. See, e.g., Avan et al., 2014 Chemical SocietyReviews, 43: 3575-3594, incorporated herein by reference. Generally,peptidomimetics are structurally similar to a paradigm polypeptide(i.e., a polypeptide that has a biochemical property or pharmacologicalactivity), such as human antibody, but have one or more peptide linkagesoptionally replaced by a linkage selected from the group consisting of:—CH₂NH—, —CH₂S—, —CH₂—CH₂—, —CH═CH— (cis and trans), —COCH₂—,CH(OH)CH₂—, and —CH₂SO—, by methods well known in the art. Systematicsubstitution of one or more amino acids of a consensus sequence with aD-amino acid of the same type (e.g., D-lysine in place of L-lysine) maybe used to generate more stable peptides. In addition, constrainedpeptides comprising a consensus sequence or a substantially identicalconsensus sequence variation may be generated by methods known in theart (Rizo and Gierasch Ann. Rev. Biochem. 61:387 (1992)); for example,by adding internal cysteine residues capable of forming intramoleculardisulfide bridges which cyclize the peptide.

As described herein, in some aspects, the peptides described herein aremodified. For example, in some cases, the conventional peptide bond(i.e., the chemical bond foliated between two molecules when thecarboxyl group of one molecule reacts with the amino group of the othermolecule, releasing a molecule of water (H₂O)) between two amino acidsis changed to a different linkage to render the resulting molecule morestable in biological fluids.

In some cases, the peptide is a cyclic peptide, wherein the aminotermini and carboxyl termini, amino termini and side chain, carboxyltermini and side chain, or side chain and side chain are linked with acovalent bond that generates a ring. Cyclic peptides are extremelyresistant to the process of digestion, enabling them to survive in thehuman digestive tract. This trait makes cyclic peptides attractive forprotein-based drugs are delivered orally.

In other cases, the amino acids of the peptides described herein aremodified. For example, modification may involve deletion of amino acids,chemical modification of certain amino acids (for example, amidation,acetylation, phosphorylation, glycosylation, formation of pyroglutamate,oxidation/reduction of sulfa group on a methionine, or addition ofsimilar small molecules) to certain amino acids.

In some cases, the Tmem 100 mutant polypeptides described hereincomprise one or more lipid modifications. At least five different typesof lipids can be covalently attached to proteins: fatty acids,isoprenoids, sterols, phospholipids, and glycosylphosphatidyl inositol(GPI) anchors. See, e.g., M. Resh, 2013 Current Biology, 23(10):pR431-pR435, incorporated herein by reference. Proteins can contain morethan one type of lipid, e.g., myristate+palmitate,palmitate+cholesterol, or famesyl+palmitate. Each type of lipid moietyis attached by a different lipid transferase and each modificationconfers distinct properties to the modified protein. The most commonoutcome of lipid modification is an increased affinity for membranes.However, attachment of myristoyl or prenyl groups can also promoteintramolecular and intermolecular protein-protein interactions. Anotherkey concept is reversibility. The covalent linkage between a protein andeither thioester-linked palmitate or a GPI anchor can be broken by theactions of thioesterases and phospholipases, respectively. By contrast,neither myristate nor the isoprenoids farnesyl or geranylgeranyl arephysically removed from a modified protein.

Optionally, the Tmem 100 mutant polypeptides described herein compriseany lipid modification, e.g., palmitoyl (Pal) or myristyl (Myr) groups,to make the peptides cell permeable. Suitable locations for modificationwith lipophilic groups include the N-terminus and/or C-terminus of thepeptide; however, the peptide may be lipid modified at any amino acidposition within the peptide.

By “isolated nucleic acid” is meant a nucleic acid that is free of thegenes which flank it in the naturally-occurring genome of the organismfrom which the nucleic acid is derived. The term covers, for example:(a) a DNA which is part of a naturally occurring genomic DNA molecule,but is not flanked by both of the nucleic acid sequences that flank thatpart of the molecule in the genome of the organism in which it naturallyoccurs; (b) a nucleic acid incorporated into a vector or into thegenomic DNA of a prokaryote or eukaryote in a manner, such that theresulting molecule is not identical to any naturally occurring vector orgenomic DNA; (c) a separate molecule such as a cDNA, a genomic fragment,a fragment produced by polymerase chain reaction (PCR), or a restrictionfragment; and (d) a recombinant nucleotide sequence that is part of ahybrid gene, i.e., a gene encoding a fusion protein. Isolated nucleicacid molecules according to the present invention further includemolecules produced synthetically, as well as any nucleic acids that havebeen altered chemically and/or that have modified backbones. Forexample, the isolated nucleic acid is a purified cDNA or RNApolynucleotide. Isolated nucleic acid molecules also include messengerribonucleic acid (mRNA) molecules.

As used herein, the terms “TRPA1”, “TRPA1 protein”, and “TRPA1 channel”are used interchangeably throughout the application. These terms referto an ion channel (e.g., a polypeptide) comprising the amino acidsequence set forth in GenBank Accession No. NP_015628 (human);Accession: NP_808449 (mouse); Accession: NP_997491 (rat), or anequivalent polypeptide, or a functional bioactive fragment thereof.

As used herein, the term “Tmem100” (or Pirt2) refers to a membraneprotein that functions as a regulator of TRPA1. In certain cases,Tmem100 refers to a polypeptide comprising, consisting of, or consistingessentially of, the amino acid sequence set forth in SEQ ID NO: 1(GenBank Ref N. NP_001093110; shown below) or SEQ ID NO: 2 (GenBank RefNo. NP_080709, shown below):

(Homo sapiens) SEQ ID NO: 1   1mteepikeil gapkahmaat mekspksevv ittvplvsei qlmaatggte lscyrciipf  61avvvfiagiv vtavaysfns hgsiisifgl vvlssglfll assalcwkvr qrskkakrre 121sqtalvanqr slfa (Mus musculus) SEQ ID NO: 2   1mteestkenl gapksptpvt meknpkrevv vttgplvsev qlmaatggae lscyrciipf  61avvvfitgiv vtavaysfns hgsiisifgl vllssglfll assalcwkvr qrnkkvkrre 121sqtalvvnqr clfa

Tmem100 includes polypeptides that retain a function of Tmem100 andcomprise (i) all or a portion of the amino acid sequence set forth inSEQ ID NO: 1 or SEQ ID NO: 2; (ii) the amino acid sequence set forth inSEQ ID NO: 1 or SEQ ID NO: 2 with 1 to about 2, 3, 5, 7, 10, 15, 20, 30,50, 75 or more conservative amino acid substitutions; (iii) an aminoacid sequence that is at least 70%, 75%, 80%, 90%, 95%, 96%, 97%, 98%,or 99% identical to SEQ ID NO:1 or SEQ ID NO: 2; and (iv) functionalfragments thereof. Polypeptides of the disclosure also include homologs,e.g., orthologs and paralogs, of SEQ ID NO: 1 or SEQ ID NO: 2.

The term “Tmem100 mutant” is meant to refer to polypeptide comprisingone or more alterations in SEQ ID NO: 1 or SEQ ID NO: 2, or fragmentsthereof. An alteration can be one or more amino acid additions,deletions or substitutions.

A Tmem100 mutant comprises a polypeptide comprising, consisting of, orconsisting essentially of, the amino acid sequence set forth in SEQ IDNO: 5 or SEQ ID NO: 6, or fragments thereof, as shown below:

(Homo sapiens) SEQ ID NO: 5MTEEPIKEILGAPKAHMAATMEKSPKSEVVITTVPLVSEIQLMAATGGTELSCYRCIIPFAVVVFIAGIVVTAVAYSFNSHGSIISIFGLVVLSSGLFLLASSALCWKVRQRSKKAQQQESQTALVANQRSLFA (Mus musculus) SEQ ID NO: 6MTEESTKENLGAPKSPTPVTMEKNPKREVVVTTGPLVSEVQLMAATGGAELSCYRCIIPFAVVVFITGIVVTAVAYSFNSIIGSIISIFGLVLLSSGLFLLASSALCWKVRQRNKKVQQQESQTALVVNQRCLFA

A Tmem100 mutant comprises a polypeptide comprising, consisting of, orconsisting essentially of, the amino acid sequence set forth in SEQ IDNO: 7 or SEQ ID NO: 8, or fragments thereof, as shown below. Preferably,these Tmem 100 mutant polypeptides comprise palmitoyl (Pal) or myristyl(Myr) groups at their N-terminus to make them cell permeable:

(Homo sapiens) SEQ ID NO: 7 WKVRQRSKKAQQQESQTALVANQRSLFA (Mus musculus)SEQ ID NO: 8 WKVRQRNKKVQQQESQTALVVNQRCLFA

The term “Tmem100” (or TMEM100) further refers to a nucleic acidencoding a polypeptide of the invention, e.g., a nucleic acid comprisinga sequence consisting of, or consisting essentially of, thepolynucleotide sequence set forth in SEQ ID NO: 3 (GenBank Ref. No.NM_001099640, shown below) or SEQ ID NO: 4 (GenBank Ref. No. NM_026433,shown below):

(Homo sapiens) SEQ ID NO: 3    1cagttgcttc tacaaaaccc gtgaaagttc tctgtccaaa agccttgttg gtcccgcggt   61acatgcttcc tgttcccaga gagattcacc cttgggcttt cctatcagtc ttccctaaag  121ttggctgctc ctgtgtcctg tcacataaaa ctgtgaaccg aggtctccga cttacgtcat  181gtcagtcaca gcagggtgag gctccacaag tgtgagttct ggcccctgct gctttccttt  241caaatgcagt ttacagttta ttatggtatt ggacacccca tgctccttac tgcattggct  301ttgggtaaga aggagtgaaa attagtgtgc gaacctgaaa acctagaatt tctgattggg  361actgaagaaa acctttgtgc tgcagtcagt ccccgagcca gacgcctgtg actctcttca  421catggaaata gatgactatc cacagtaaaa gcaaaattaa aaagtgactt gtgaaaaata  481tcctccatac tcctcttacc aagcctgcaa cttaaaacct atggtttaaa ctgtgctttc  541aattatctgg aggaggccag cactgatgag cccatcctga gccagtcatt ttaaggccag  601tgctacctaa ctgagacaag gctaatctgg tcgccttgct gttagaggct ctttcccaga  661agttggacga agaggctcag gcgttgctgt ttcttgtctt ccaagtcaag tggttactct  721ggtaatggat tgcctctctc cgagctttca ccctggtgag actgtccaga tctagtctgt  781aaacccagct tagaagcact gttgtaaaaa tgactgaaga gcccatcaag gagatcctgg  841gagccccaaa ggctcacatg gcagcgacga tggagaagag ccccaagagt gaagttgtga  901tcaccacagt ccctctggtc agtgagattc agttgatggc tgctacaggg ggtaccgagc  961tctcctgcta ccgctgcatc atcccctttg ctgtggttgt cttcatcgcc ggcatcgtgg 1021tcaccgcggt ggcttacagc ttcaattccc atgggtctat tatctccatc tttggcctgg 1081ttgttctgtc atctggactt tttttactag cctccagtgc cttgtgctgg aaagtgagac 1141aaaggagcaa gaaagccaag agacgggaga gtcaaacagc tctcgtggca aatcagagaa 1201gcttgtttgc ttgagactga atacgaccaa atgggccatt gggcctggaa aacgtgctct 1261gactttgtca cccaattcac ccagaaccat ggtgggagag aacagacttg gcgttggagc 1321agactggaag aatgggggtg ggagggtgga ggggcttctc ctttgtgagg aatgactcat 1381gtcttcttta acgacaaact taaccctaag ggctacttct gagactgaaa aatcagcttt 1441ctatttacat gaaacacttt gggggtcatg gaagtgcaca gcattagaca gtatttggtt 1501caccctgtaa agtagccaag aaaagatgag aaaaatcaag ataggcctgg cacactagac 1561atttgcctcc aaaagaaata acctacagtc ttaagatgta tcataaaaat gttctgccaa 1621ggatctaaat taccttgggt ttcgcatatg tctatgaaat tctgtgataa tttttttcaa 1681tacattgatt cactggcgtc tgttttcatt ttatactttt aataactcat cactggtggt 1741actttatctt gaaaagtaat attttttata ttttaacatt ggacagtgtt agccagttgt 1801aatgatgtat cagaagtaaa gaaaaaccca ttaaagttat agctaataga tgctgttggg 1861ggttaaatta atagtaaaat aatccaatat agcacttttg atgattttta tataaaagtc 1921aactgtacat ttcattcaga ataataaata cttattgctg ctaaaacttc ttaaatggtt 1981gtttctgcta tagttatttc tattgcagtt ccaaattgcc atcttccctt gtctcatttg 2041caagttctca attgtatttc tctcaaatgg acaggttcct tctttactgg aggatttttg 2101tttttatcat attggttttt cattacttct gaatagtctt aattacgttt actaaattct 2161aaaggatttc tgtgctatta taattaggaa atcaacgtct ttggtcagga actttataat 2221gtgctattaa atgtatatta catttttgtg gaaaaaaaaa aaaaaaaaa (Mus musculus)SEQ ID NO: 4    1tctgcagctt cacactacaa aagagtgata tcgaagacta ggttagagaa aaccataaaa   61aagaatgctt gcataaggat tttaaacggg ggaaagaaaa caggagagat ttttagtgac  121tttttaaata attgggttat gatggattca ctcctgtaat tacagtggat ggtgtgggca  181cggaaaaaaa aatagaaaag aaaaggaaaa cgtagagtaa aatacaacag cgagtaagga  241atttcctttc accaaattgt ctccggtccc ttaatggtct gtaaatcttg ctgagtactt  301tgtgtacggt ccctagcatg gtgatttgca tcccactgtc ggcctcgctg ttggccgccg  361cttgctgctg tctcagtcca cttctggctg agaaagaggc aatccctggt ctgtcctttt  421accaatgccc agtgggtgac aggctctttc ccagaagttg aacggagcta cgctggcaga  481tccctctctc ccaagtcaag tggcctctct ggtaatggac tgcctttctg tgagcttgca  541tcctgaccag gctttccaaa tctagcctgt gaagcgagat aagaaaaatc ccacagaaga  601aaaaaaaatg accgaagaat ccacaaaaga gaacctggga gctccaaaat ctcccacacc  661tgtgacaatg gagaaaaacc ccaagaggga agttgtggtc accacgggac ccttggtcag  721cgaggttcag ctgatggccg ccaccggggg tgccgaactc tcctgctacc gctgcatcat  781cccctttgcc gtggtggtct tcatcactgg gattgtggtc accgctgtgg cttacagctt  841caattcccat ggttccatca tctccatctt cggcctggtc cttctgtcct ccggactgtt  901tttactagcc tccagtgcct tgtgctggaa ggtgagacaa aggaacaaga aagtcaagag  961acgcgagagt cagaccgctc ttgtggtaaa tcagaggtgc ttgtttgctt aagactgagt 1021aagagcaaac gcgaacgctc acccgcccac actgctctaa ctcagtgaac tcattcatag 1081gaaaccagcc cggtaccgga tgagctcaac tttcgaatga actaccaaaa tgaaaaatag 1141ggcatagaga ggactgaaaa tgaggggtca agacggttcc cccttggtag ggaataactc 1201atctttaacg acaaacttaa ccctaagggc tatttctaac acagacagag gaatcaactt 1261gcttttcctg ttaaacgttt agggagtgtg ggagatgcac agcattaaat aacagttggt 1321tccatttaga aagtacccaa gggaagaatg gacaaataat gagagccctg gcaagtggtg 1381ttataaaaac gttccaccaa aagcctacat tggcttggca ttcccacgta cctaagaagt 1441tctgttatat atatacatat atattttttc caataagttg attctttgcc cccccctttt 1501taaaagaatt ttcactttca gtaacatcac tagaggtact ttattttgaa gaatagacta 1561atatttttta tattttaaca atggacaatt gtagatggtt gtaatgatat gtcagaagaa 1621aacagaaatg taggtaacac agatgacaca gggacagtta aattaatatt gaaataatcc 1681aatctagcac ctttgatggc ttttatacaa aagttcagtg tgcatttcac tcaaaataat 1741aaatgctcat ggctgctgaa actt

The term “Tmem100 mutant” refers to a nucleic acid encoding apolypeptide of the invention, e.g., a nucleic acid comprising a sequenceconsisting of, or consisting essentially of, the polynucleotide sequenceset forth in SEQ ID NO: 9 or SEQ ID NO: 10.

(Homo sapiens) SEQ ID NO: 9ATGACTGAAGAGCCCATCAAGGAGATCCTGGGAGCCCCAAAGGCTCACATGGCAGCGACGATGGAGAAGAGCCCCAAGAGTGAAGTTGTGATCACCACAGTCCCTCTGGTCAGTGAGATTCAGTTGATGGCTGCTACAGGGGGTACCGAGCTCTCCTGCTACCGCTGCATCATCCCCTTTGCTGTGGTTGTCTTCATCGCCGGCATCGTGGTCACCGCGGTGGCTTACAGCTTCAATTCCCATGGGTCTATTATCTCCATCTTTGGCCTGGTTGTTCTGTCATCTGGACTTTTTTTACTAGCCTCCAGTGCCTTGTGCTGGAAAGTGAGACAAAGGAGCAAGAAAGCCCAGCAACAGGAGAGTCAAACAGCTCTCGTGGCAAATCAGAGAAG CTTGTTTGCTTGA(Mus musculus) SEQ ID NO: 10ATGACCGAAGAATCCACAAAAGAGAACCTGGGAGCTCCAAAATCTCCCACACCTGTGACAATGGAGAAAAACCCCAAGAGGGAAGTTGTGGTCACCACGGGACCCTTGGTCAGCGAGGTTCAGCTGATGGCCGCCACCGGGGGTGCCGAACTCTCCTGCTACCCTCTGCATCATCCCCTTTGCCGTGGTGGTCTTCATCACTGGGATTGTGGTCACCGCTGTGGCTTACAGGTTCAATTCCCATGGTTCCATCATCTCCATCTTCGGCCTGGTCCTTCTGTCCTCCGGACTGTTTTTACTAGCCTCCAGTGCCTTGTGCTGGAAGGTGAGACAAAGGAACAAGAAAGTCCAGCAACAGGAGAGTCAGACCGCTCTTGTGGTAAATCAGAGGT GCTTGTTTGCTTAA

The term “Tmem100 mutant” refers to a nucleic acid encoding apolypeptide of the invention, e.g., a nucleic acid comprising a sequenceconsisting of, or consisting essentially of, the polynucleotide sequenceset forth in SEQ ID NO: 11 or SEQ ID NO: 12.

(Homo sapiens) SEQ ID NO: 11TGGAAAGTGAGACAAAGGAGCAAGAAAGCCCAGCAACAGGAGAGTCAAACAGCTCTCGTGGCAAATCAGAGAAGCTTGTTTGCTTGA (Mus musculus) SEQ ID NO: 12TGGAAGGTGAGACAAAGGAACAAGAAAGTCCAGCAACAGGAGAGTCAGACCGCTCTTGTGGTAAATCAGAGGTGCTTGTTTGCTTAA

A nucleic acid of the disclosure can comprise all, or a portion of: thenucleotide sequence of SEQ ID NO: 3, 4, 9, 10. 11 or 12; a nucleotidesequence at least 70%, 75%, 80%, 90%, 95%, 96%, 97%, 98% or 99%identical to SEQ ID NO: 3, 4, 9, 10, 11 or 12; a nucleotide sequencethat hybridizes under stringent conditions to SEQ ID NO: 3, 4, 9, 10, 11or 12; nucleotide sequences encoding polypeptides that are functionallyequivalent to polypeptides of the disclosure; nucleotide sequencesencoding polypeptides at least about 70%, 75%, 80%, 85%, 90%, 95%, 98%,99% homologous or identical with an amino acid sequence of SEQ ID NO: 3,4, 9 or 10; nucleotide sequences encoding polypeptides having anactivity of a polypeptide of the disclosure and having at least about70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or more homology or identity withSEQ ID NO: 3, 4, 9, 10, 11 or 12; nucleotide sequences that differ by 1to about 2, 3, 5, 7, 10, 15, 20, 30, 50, 75 or more nucleotidesubstitutions, additions or deletions, such as allelic variants, of SEQID NO: 3, 4, 9, 10, 11 or 12; nucleic acids derived from andevolutionarily related to SEQ ID NO: 3, 4, 9, 10, 11 or 12; andcomplements of, and nucleotide sequences resulting from the degeneracyof the genetic code, for all of the foregoing and other nucleic acids ofthe invention. Nucleic acids of the disclosure also include homologs,e.g., orthologs and paralogs, of SEQ ID NO: 3, 4, 9 or 10 and alsovariants of SEQ ID NO: 3, 4, 9, 10, 11 or 12 which have been codonoptimized for expression in a particular organism (e.g., host cell).Where not explicitly stated, one of skill in the art can readily assesswhether Tmem100 refers to a nucleic acid or a protein.

Tmem100 weakens the association of TRPA1 and TRPV1 and thereby releasesthe inhibition of TRPA1 by TRPV1. A “Tmem100 mutant” exerts the oppositeeffect, i.e. it enhances the association of TRPA1 and TRPV1 and inhibitsTRPA1.

As used herein, the terms “prevent,” “preventing,” “prevention,”“prophylactic treatment,” and the like, refer to reducing theprobability of developing a disease or condition in a subject, who doesnot have, but is at risk of or susceptible to developing a disease orcondition.

The term “treating” includes prophylactic and/or therapeutic treatments.The term “prophylactic or therapeutic” treatment is art-recognized andincludes administration to the host of one or more of the subjectagents. If it is administered prior to clinical manifestation of theunwanted condition (e.g., disease or other unwanted state of the hostanimal) then the treatment is prophylactic, (i.e., it protects the hostagainst developing the unwanted condition), whereas if it isadministered after manifestation of the unwanted condition, thetreatment is therapeutic, (i.e., it is intended to diminish, ameliorate,or stabilize the existing unwanted condition or side effects thereof).

Ranges provided herein are understood to be shorthand for all of thevalues within the range. For example, a range of 1 to 50 is understoodto include any number, combination of numbers, or sub-range from thegroup consisting 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50.

The recitation of a listing of chemical groups in any definition of avariable herein includes definitions of that variable as any singlegroup or combination of listed groups. The recitation of an embodimentfor a variable or aspect herein includes that embodiment as any singleembodiment or in combination with any other embodiments or portionsthereof.

Any composition, agents or methods provided herein can be combined withone or more of any of the other compositions, agents and methodsprovided herein.

Other features and advantages of the invention will be apparent from thefollowing description of the preferred embodiments thereof, and from theclaims. Unless otherwise defined, all technical and scientific termsused herein have the same meaning as commonly understood by one ofordinary skill in the art to which this invention belongs. Althoughmethods and materials similar or equivalent to those described hereincan be used in the practice or testing of the present invention,suitable methods and materials are described below. All publishedforeign patents and patent applications cited herein are incorporatedherein by reference. Genbank and NCBI submissions indicated by accessionnumber cited herein are incorporated herein by reference. All otherpublished references, documents, manuscripts and scientific literaturecited herein are incorporated herein by reference. In the case ofconflict, the present specification, including definitions, willcontrol. In addition, the materials, methods, and examples areillustrative only and not intended to be limiting.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A-FIG. 1F demonstrate that Tmem100 is expressed in TRPA1/TRPV1positive peptidergic DRG neurons. FIG. 1A is a schematic drawing of thestructure of Tmem100. Tmem100 is a two-transmembrane protein with aputative TRPA1 binding site (KRR) at its C-terminus. Both the N- andC-termini are intracellular whereas the loop region is extracellular.“PM”: plasma membrane. FIG. 1B is a graph showing thatTmem100-expressing cells comprise 24% of L4-L6 DRG neurons. They arepredominantly small-diameter, but medium- to large-diameter neurons arealso present. The average diameter of Tmem100-expressing neurons is 15.7μm, and the median is 14.3 μm (DRG from 3 mice). FIG. 1C shows images ofdouble staining of Tmem100-GFP with other DRG markers. Bar: 50 μm. FIG.1D is a table of the quantification of co-expression of Tmem100 andother DRG markers (DRG from 3 mice; data are presented as mean±SEM).FIGS. 1E and 1F are diagrams showing the relationship of Tmem100 withother DRG markers. Tmem100 is a marker for the majority of CGRP⁺ DRUneurons (FIG. 1E); most TRPA1⁺ DRG neurons express Tmem100 (FIG. 1F).

FIG. 2A-FIG. 2J demonstrate that Tmem100 CKO mice show selectivedeficits in TRPA1-associated behaviors while TRPV1-associated behaviorsare unaffected. FIG. 2A is a schematic depiction of the Tmem100conditional KO strategy. FIG. 2B shows images of the deletion of theTmem100 gene in conditional KO lines was verified by anti-Tmem100immunostaining of DRG. Bar: 50 μm. FIG. 2C is a graph of the baselinemechanical sensitivity of Tmem100 CKO (Avil-Cre;Tmem100^(fl/fl)) mice issimilar to that of the control group (Avil-Cre;Tmem100^(+/+) mice). Atthe forces tested, there was no significant difference in the responserate among Tmem100 CKO and the control group (n=10 for CKO and 13 forcontrol). FIG. 2D is a graph indicating that Tmem100 CKO mice showeddecreased acute nocifensive behavior after 0.2% MO injection. (Whitebar: Avil-Cre;Tmem100^(+/+); grey bar: Tmem100^(fl/fl); black bar:Avil-Cre;Tmem100^(fl/fl). Total time of licking and flinching: 86±6.2sec in Avil-Cre;Tmem100^(+/+); 86±11.1 sec in Tmem100^(fl/fl); 57±9.3sec in Tmem100 CKO; n=13 for Avil-Cre;Tmem100^(+/+) and Tmem100^(fl/fl);n=10 for Tmem100 CKO) *p<0.05, **p<0.01. FIG. 2E is a graph indicatingthat Tmem100 CKO mice showed decreased mechanical hyperalgesia after MOinjection (n=11 for Avil-Cre;Tmem100^(+/+); 9 for Tmem100^(fl/fl); 10for Tmem100 CKO). FIG. 2F is a graph showing Capsaicin-inducednocifensive behaviors (0.6 μg) are similar between Tmem100 CKO and thecontrol group (Avil-Cre;Tmem100^(+/+) mice). Time of licking andflinching: 45±5.7 sec in Avil-Cre; Tmem100^(+/+) v.s 53.7±8.3 sec inTmem100 CKO; n=12 in CKO and 10 in control; p=0.40). FIGS. 2G and 2H aregraphs indicating that Tmem100 CKO mice show decreased mechanicalhyperalgesia but intact thermal hyperalgesia after CFA injection. Oneand 2 days after CFA injection, Tmem100 CKO mice had higher mechanicalthresholds than both control groups, but the response to painful thermalstimuli remained unchanged. (*p<0.05; **p<0.01; ***p<0.001; n=16 forAvil-Cre;Tmem100^(+/+); 10 for Tmem100^(fl/fl); 18 for Tmem100 CKO for(FIG. 20); n=9 for Avil-Cre;Tmem100^(+/+); 10 for Tmem100^(fl/fl); 11for Tmem100 CKO for (FIG. 2H)). FIGS. 2I and 2J are graphs indicatingthat nocifensive responses in the hot plate and tail immersion testswere intact in Tmem100 CKO mice. At the indicated temperatures, therewas no difference in the latency between CKO and control groups(Avil-Cre;Tmem100^(+/+) mice) (n=9 and 13 for control and CKO,respectively, in (FIG. 2I); n=13 and 10 for control and CKO,respectively, in (FIG. 2J)). All statistics are unpaired t-tests exceptfor (FIG. 2C), in which two-way ANOVA with Bonferroni's post-hoc test isused. Data are presented as mean±SEM.

FIG. 3A-FIG. 3G indicate that Tmem100 enhances TRPA1-mediated responsesin a TRPV1-dependent manner. FIG. 3A is a graph showing that TRPA1activity in the DRG neurons of Tmem100 CKO mice was reduced whereasTRPV1 activity was relatively unchanged. The percentage of neuronsresponding to 10 μM mustard oil (MO) and 250 μM cinnamaldehyde (CA) waslower in the CKO line whereas the percentage of neurons responding to100 nM capsaicin (CAP) and 100 μM menthol were similar between the CKOand control groups. (Each reagent was tested greater than 3 times in 3mice; greater than 300 IB4⁻ cells were assessed in the MO, CA, and CAPgroup and greater than 297 cells were assessed in the MEN group. Allstatistics are unpaired t-tests. The error bars represent SEM; *p<0.05;**p<0.01) FIG. 3B is a graph showing that the average current induced by7 or 25 μM MO (1 min application) is lower in DRG neurons from Tmem100CKO mice, whereas there is no significant difference when 150 μM MO isapplied. The numbers in the columns represent the number of cellsresponding/the number of cells tested. More than 3 mice were tested fromeach group; all statistics are an unpaired t-tests. All error barsrepresent SEM; **p<0.01). FIG. 3C shows representative traces ofMO-induced currents from Avil-Cre;Tmem100^(+/+) andAvil-Cre;Tmem100^(fl/fl) mice. FIG. 3D is a graph indicating the averagecurrent induced by 100 nM CAP (30 sec application) is similar among DRGneurons from Tmem100 CKO and control mice. The numbers in the columnsrepresent the number of cells responding/the number of cells tested.More than 3 mice were tested from each group; all statistics areunpaired t-tests. The error bars represent SEM. FIG. 3E showsrepresentative traces of CAP-induced currents fromAvil-Cre;Tmem100^(+/+) and Avil-Cre;Tmem100^(fl/fl) mice. FIG. 3F is agraph showing MO (10 μM)-gated whole-cell voltage clamp (Vh=−60 mV)current densities in TRPA1+TRPV1 (1:1)- and TRPA1+TRPV1+Tmem100(1:1:4)-expressing CHO cells. The number of cells analyzed and thosethat responded are indicated within bars. The statistic is an unpairedt-test (**p<0.01). The error bars represent SEM. FIG. 3G is a graphshowing MO (10 μM)-evoked Ca²⁺ influx into TRPA1+TRPV1 (1:1)- andTRPA1+TRPV1+Tmem100 (1:1:4)-expressing CHO cells. The numbers of cellsresponding are indicated within bars. The statistic is an unpairedt-test (***p<0.001). Data are presented as mean±SEM.

FIG. 4A-FIG. 4E demonstrate context-dependent regulation of Tmem100 inthe TRPA1-V1 complex. FIGS. 4A and 4B are graphs showing MO (10 μM)- (A)and CAP (100 nM)-induced (B) single-channel open probability (P_(o)) ofthe main conductance at Vh=−60 mV in CHO cells expressing TRPA1 vs.TRPA1+Tmem100 (T100) (1:4 molar ratio) and TRPA1+TRPV1 (1:1) vs.TRPA1+TRPV1+Tmem100 (1:1:4). The configuration is cell-attached patch,and the number of cells that responded among those analyzed is indicatedwithin bars. The statistic is one-way ANOVA with Bonferroni's post-hoctest for comparison between all columns (**p<0.01; ***p<0.001); errorbars represent SEM. FIGS. 4C and 4D show representative single-channelrecording during 5 sec at Vh=−60 mV for MO-gated current (FIG. 4C) and 4sec for CAP-gated current (FIG. 4D) in TRPA1+TRPV1- andTRPA1+TRPV1+Tmem100-expressing CHO cells; c is the closed state, o1, o2,and o3 show open states for 3 independent channels in the patch. FIG. 4Eis a table of summary of data on single-channel conductance (pS) andeffects on Po changes of Tmem100 (T100) for the given agonists and genesexpressed. The values of single-channel conductance were derived fromFIG. 12A-12K. Data are presented as mean±SEM.

FIG. 5A-FIG. 5F demonstrate that Tmem100 decreases the interactionbetween TRPA1 and TRPV1 whereas the Tmem100-3Q mutant enhances it. FIG.5A is an image of a Co-IP with Tmem100 and TRPV1 antibodies and Westernblotting with TRPA1, TRPV1, and Tmem100 antibodies in mouse DRG lysates.Tmem100, TRPA1, and TRPV1 form a complex in DRG neurons. FIG. 5 is animage of a Co-IP of TRPV1 and full-length myc-Tmem100 inTRPV1-expressing cells. The results show an interaction between Tmem100and TRPV1. FIG. 5C is an image of a Co-IP of TRPA1 and full-lengthmyc-Tmem100 in TRPA1-V1 co-expressing cells. Tmem100 binds TRPA1 atdifferent dilutions. FIG. 5D is an image of a GST pull-down withdifferent TRPs and fragments of Tmem100 and Tmem100-3Q proteins. TRPV1is pulled down by the C-termini of both WT Tmem100 (GST-C-WT) andTmem100-3Q (GST-C-3Q). It is also pulled down by the N-terminus of WTTmem100 (GST-N). However, TRPA1 is pulled down only by the C-terminus ofWT Tmem100, whereas the Q-Q-Q mutation abolishes this interaction. Thelysates from the cells expressing unconjugated GST (GST) and each TRPserve as negative controls. FIG. 5E is a graph showing FRET results withTIRF microscopy for the effects of Tmem100 on TRPA1-V1 interactions. Allgroups were transfected with TRPV1-CFP and TRPA1-YFP except for theRho-pYC (as a positive control for maximal effects of the system) andM-V1 (negative control) groups, where Rho-pYC and membrane-tetheredYFP+TRPV1-CFP, respectively, were transfected instead. FRET efficiencieswere highest in Tmem100-3Q-transfected cells (A1-V1-3Q), followed by theempty vector-transfected groups (A1-V1), and were lowest in cellstransfected with wild-type Tmem100 (A1-V1-WT). The statistic is one-wayANOVA (*p<0.05; **p<0.01, ***p<0.001). Data are presented as mean±SEM.FIG. 5F is a table showing a summary of the data from FIG. 5E.

FIG. 6A-FIG. 60 demonstrate context-dependent regulation of Tmem100-3Qmutant in the TRPA1-V1 complex. FIGS. 6A and 6B are graphs showing MO(10 μM)-(FIG. 6A) and CAP (100 nM)-induced (FIG. 6B) single-channel openprobability (P_(o)) of the main conductance at Vh=−60 mV in CHO cellsexpressing TRPA1 vs. TRPA1+Tmem100-3Q (T100-3Q) (1:4 molar ratio) andTRPA1+TRPV1 (1:1) vs. TRPA1+TRPV1+Tmem100-3Q (1:1:4). The statistic isone-way ANOVA as in FIG. 4; *p<0.05; **p<0.01; NS: no significantdifference. FIGS. 6C and 6D show representative single-channel recordingtraces at Vh=−60 mV for MO- (FIG. 6C) and CAP-gated (FIG. 6D) current inTRPV1+TRPA1- vs. TRPV1+TRPA1+Tmem100-3Q-expressing CHO cells. Traces are4 sec long. FIG. 6E is a table summary of data on single-channelconductance (pS) and effects on P_(o) changes of Tmem100-3Q for thegiven agonists and genes expressed. The values of single-channelconductance were derived from FIG. 12. FIG. 6F is a graph showing MO (10μM)-gated whole-cell voltage clamp (Vh=−60 mV) current densities inTRPA1+TRPV1 (1:1)- and TRPA1+TRPV1+Tmem100-3Q (1:1:4)-expressing CHOcells. Results: 138.9±29.92 pA/pF for TRPA1+TRPV1 vs. 39.16±12.5 pA/pFfor TRPA1+TRPV1+Tmem100-3Q cells. The statistic is an unpaired t-test(**p<0.01). FIG. 6G is a graph showing MO (10 μM)-evoked Ca²⁺ influxinto TRPA1+TRPV1 (1:1)- and TRPA1+TRPV1+Tmem100-3Q (1:1:4)-expressingCHO cells. Results: 463.1±26.8 nM for TRPA1+TRPV1 vs. 321±18.2 nM forTRPA1+TRPV1+Tmem100-3Q cells. The numbers of cells responding/tested areindicated within bars. The statistic is an unpaired t-test (***p<0.001).Data are presented as mean±SEM.

FIG. 7A-FIG. 7I demonstrate that T100-Mut cell-permeable peptide(T100-Mut) alleviates TRPA1-associated pain. FIG. 7A shows sequences ofthe cell-permeable peptide T100-Mut (SEQ ID NO: 8), scrambled peptide(SEQ ID NO: 15), T100-WT (SEQ ID NO: 14), and WT (SEQ ID NO: 13)C-terminal sequence of Tmem100 (WT). Myr: myristoylation. FIG. 7B is agraph of calcium imaging data from the T100-Mut-treated (200 nM) DRGneurons. (MO: 12±1.3% in scrambled vs. 4.6±0.9% in T100-Mut-treatedgroup; CAP: 16±1.3% in scrambled vs. 15±6.5% in T100-Mut; DRG from 3mice). FIG. 7C is a graph of calcium imaging data from HEK293T cellsexpressing TRPA1 and TRPV1. Pre-treatment of T100-Mut (200 nM) reducedthe percentage of cells responsive to 500 nM MO, whereas T100-WTproduced no such effect (18±2.8% in scrambled, 6±2.4% in T100-Mut, and17±0.6%, repeated 3 times, *p<0.05). FIG. 7D is a graph of pre-treatmentwith T100-Mut (5 μl of 2 mM) alleviated MO-induced acute nocifensivebehavior (n=6, **p<0.01) and mechanical hyperalgesia (0.1±0.03 g inscrambled vs. 0.4±0.11 g in T100-Mut; n=13, *p<0.05). FIG. 7E is a graphof pre-treatment with T100-Mut (5 μl of 2 mM) alleviated CFA-inducedmechanical hyperalgesia (0.05±0.0 g in scrambled vs. 0.43±0.1 g inT100-Mut; n=10, ***p<0.001) but not thermal hyperalgesia (3.9±0.6 sec inscrambled vs. 4.3±0.5 sec in T100-Mut; n=10, p=0.63). FIG. 7F is a graphshowing acute nocifensive behaviors induced by intradermal capsaicininjection (0.6 μg) were similar among the T100-Mut- and scrambledpeptide-treated (2 mM) WT mice (70±11 sec in scrambled vs. 65±9 sec inT100-Mut; n=8, p=0.73). FIG. 7G is a graph showing that in wild-typemice injected with paclitaxel (Taxol), mechanical hyperalgesia wasobserved at day 7 post-injection. Hyperalgesia was attenuated by theintradermal injection of T100-Mut (n=7 for scrambled control peptide(open bar); n=8 for T100-Mut peptide (black bar); *p<0.05). FIGS. 7H and7I showing that in TRPV1^(−/−) mice, T100-Mut did not perturb MO-inducedacute pain (FIG. 7H) and mechanical hyperalgesia (FIG. 7I), as observedin WT mice. (n=8, p=0.91 for (FIG. 7H) and 0.64 for (FIG. 7I)). Allstatistics are unpaired t-tests and data are presented as mean±SEM.

FIG. 8A-FIG. 8D illustrate a schematic model for the modulatory effectsof TRPV1 on TRPA1 alone (FIG. 8A), WT Tmem100 (FIG. 8B), Tmem100-3Q(FIG. 8C), and T100-Mut CPP (FIG. 8D) (with myristoylated group insertedinto the plasma membrane as shown by the orange wiggly line). Thedistance between TRPA1 and TRPV1 represents the degree of association(the smaller the distance, the stronger the association). The number ofminus signs and the size of the green arrows represent the relativestrength of inhibition of TRPA1 by TRPV1.

FIG. 9A-FIG. 9I are related to FIG. 1A-FIG. 1F. FIGS. 9A, 9B and 9C areimages showing Tmem100-myc signal was only visible in the plasmamembrane when F11 cell lines transfected with a Tmem100-myc constructwere treated with detergent (FIG. 9A). Similar results were obtained inTmem100-transfected cell lines treated with an anti-Tmem100 antibodytargeting the N-terminus (FIG. 9B). No signal was detected withoutdetergent treatment (FIG. 9C). FIG. 9D shows images of a comparison ofbright field image and c-myc staining of F11 cell lines transfected withTmem100-myc suggested that Tmem100 primarily localizes to the plasmamembrane (134 total cells were analyzed). FIG. 9E is a schematic ofTmem100 GFP knock-in strategy for creation of a Tmem100^(+/GFP) mouseline. The open reading frame (CDS) of Tmem100 was replaced with atargeting construct containing farnesylated enhanced GFP and the ACEpromoter-Cre recombinase-Neomycin selection cassette. FIG. 9F showsimages of double staining of anti-GFP (green) and anti-Tmem100 (red)antibodies in Tmem100^(GFP/+) mice confirmed GFP is a surrogate markerfor Tmem100 in DRG. FIG. 9G shows images of double staining of TRPA1 andIB4 in DRG. The anti-TRPA1 antibody is specific as there is noappreciable level of background signal in DRG from TRPA1 KO mice.Moreover, TRPA1 is not expressed in IB4⁺ neurons. FIGS. 9H and 9I showthat Tmem100 is upregulated in the DRG neurons after inflammation. FIG.9H is a graph illustrating that the percentage of Tmem100-expressingneurons in L4 DRG was increased in the CFA model at days 1 and 4compared to L4 DRG neurons on the contralateral side (control). (DRGfrom 3 mice, **p<0.01 at day 1; *p<0.05 at day 4.) FIG. 9I shows imagesof immunofluorescent staining with anti-Tmem100 antibody in the L4 DRGat day 1. All data were analyzed using Student's unpaired t-test. Allbars are presented as mean±SEM.

FIG. 10A-FIG. 10I are related to FIG. 2A-FIG. 2J. FIGS. 10A-10Cdemonstrate that TRPA1 and TRPV1 levels are not altered in DRG fromTmem100 CKO mice. FIG. 10A shows images of western blots usinganti-TRPV1, -TRPA1, -Tmem100, and -actin antibodies fromAvil-Cre;Tmem100^(+/+) and Avil-Cre;Tmem100^(fl/fl) (Tmem100 CKO) mice.FIG. 10B is a graph of pooled results of TRPA1/actin band intensities.(n=8; p=0.79; 108±21 in Avil-Cre;Tmem100^(+/+) and 116±20 in Tmem100CKO). FIG. 10C is a graph of pooled results of TRPV1/actin bandintensities. (n=8; p=0.58; 38±8 in Avil-Cre;Tmem100^(+/+) and 31±9 inTmem100 CKO). FIGS. 10D-10F demonstrate that developmental andmorphological defects were not observed in Tmem100 conditional knockout(Tmem100 CKO) mice. FIG. 10D is a table quantifying immunofluorescentstaining of DRG markers in Avil-Cre; Tmem100^(GFP/fl) mice. None of themarkers tested showed evidence of cell type-specific defects in micelacking Tmem100 in their primary sensory neurons. (n>3 for each marker;8-week-old male). FIG. 10E shows images of double staining of GFP(green) and CGRP (red) in the lumbar spinal cord of adult mice. FIG. 10Fshows images of double staining of GFP (green) and IB₄ (red) in thelumbar spinal cord of adult mice. FIG. 10G is a graph showing the acutepain phenotype induced by MO is dose-dependent. N=6 for 0.02%, 13 and 11for Avil-Cre; +/+ and Avil-Cre;fl/fl for 0.2%, and 9 and 6 forAvil-Cre;+/+ and Avil-Cre;fl/fl for 2%. **p<0.01. All data were analyzedusing Student's unpaired t-test. All bars are presented as mean±SEM.FIG. 10H and FIG. 10I are graphs showing Cold-induced activities are notaltered in Tmem100-deficient mice. FIG. 10H is a graph showing thepercentage of DRG neurons activated by the temperature cooling from 22.5to 13.8° C. with bath perfusion is not significantly different fromAvil-Cre;Tmem100^(+/+) and Avil-Cre;Tmem100^(fl/fl) groups. (N=19; 16±2in Avil-Cre;Tmem100^(+/+) and 12±4 in Avil-Cre;Tmem100^(fl/fl); p=0.41).FIG. 10I is a graph showing acute pain evoked by the cold plate of zerodegree is not altered in Tmem100-deficient mice. N=7. All data wereanalyzed using student's T-test. All bars are presented as mean±SEM.

FIG. 11A-FIG. 11M are related to FIG. 3A-FIG. 3G. FIGS. 11A-11C showDose-response of MO in DRG neurons and a heterologous system. FIG. 11Ais a graph of calcium imaging of Avil-Cre; Tmem100^(+/+) and Avil-Cre;Tmem100^(fl/fl) DRG neurons with MO of different concentrations. N=3, 6,and 9 for 5, 20, and 70 μM, respectively. ***p<0.001. Student's t-testwas applied for analysis. FIG. 1B is a graph quantifying whole-cellrecording of DRG neurons with different concentration of MO (N=7, 10,12, 10, 12 and 9 for 1, 10, 25, 50, 100 and 300 μM). FIG. 11C is a graphquantifying single-channel recording of CHO cells expressing TRPA1 andTRPV1 or TRPA1 only, respectively (N=6, 7, 10, 10, 10, 9 and 9 for 0.1,1, 10, 25, 50, 100 and 300 μM). FIGS. 11D-11F demonstrate that Tmem100does not significantly affect TRPA1 and TRPV1 membrane trafficking. FIG.11D is an image of biotinylation assays of CHO cells expressingmyc-TRPA1, TRPV1, and either Tmem100 or Tmem100-3Q mutant. Beta1integrin and beta-actin were used as positive controls for thebiotinylated membrane and cytoplasmic fractions, respectively. FIG. 11Eis a graph quantifying the level of biotinylated fraction of TRPA1. TheTRPA1/beta1 integrin ratio was calculated and normalized to the ratiofrom the group transfected with myc-TRPA1 and TRPV1 only (marked asvector). Compared to the vector group, the results did not suggest asignificant difference in wild-type (WT) Tmem100-transfected (T100) orTmem100-3Q mutant-transfected cells (T100-3Q). However, the data suggesta trend towards decreased surface levels of TRPA1 in the presence ofTmem100 and increased surface levels of TRPA1 with Tmem100-3Q mutantco-expression. FIG. 11F is a graph quantifying the level of biotinylatedportion of TRPV1. The results were analyzed in the same manner as FIG.11E. The data suggest that WT Tmem100 and Tmem100-3Q mutant do not altertrafficking of TRPV1 to the surface (n=4 for (FIG. 11E) and (FIG. 11F);one-way ANOVA with Bonferroni post hoc was performed for Figures (FIG.11E) and (FIG. 11F); NS: non-significant). FIG. 11G-11M demonstrate thatTRPA1 activity evoked by MO and CA is also enhanced in the presence ofTmem100 in an alternative expression system: HEK293T cells. FIGS. 11Gand 11H are graphs indicating that TRPV1 is required for the enhancingeffect of Tmem100 on TRPA1 activity. In TRPA1−V1− expressing HEK293Tcells, Tmem100 increased the percentage of cells that respond to 100 nMMO and 5 μM CA. This enhancing effect of Tmem100 was abolished whenTRPV1 was replaced with TRPM8, showing no interaction between TRPA1 andTRPM8. The activation of TRPA1 was monitored with calcium imaging. Eachgroup was tested greater than 3 times; *p<0.05; **p<0.01. The statisticis one-way ANOVA with Bonferroni's post-hoc test. All error barsrepresent SEM. The baseline 340/380 ratios for the cells tested were notdrastically different (TRPA1+V1: 1.1±0.03; TRPA1+V1+Tmem100: 1.1±0.04;TRPA1+TRPM8+Tmem100: 0.95±0.04). FIG. 11I is a graph quantifyingsingle-channel recording of CHO cells expressing TRPA1+TRPV1 with orwithout Tmem100 at a ratio of 1:1:1. Numbers of responsive cells aremarked within bars. FIG. 11J is a graph indicating that Tmem100increases the potency of TRPA1 activity in cells expressing both TRPA1and TRPV1. In a heterologous system, Tmem100 lowered the EC₅₀ of CA(EC₅₀=21 μM in A1+V1+Tmem100 and 57 μM in A1+V1; each concentration wastested more than 3 times). *p<0.05. FIG. 11K is a graph quantifyingsingle-channel recording of the CHO cells expressing TRPA1+TRPV1 with orwithout Tmem100 under different concentration of MO. N=5-8/trial.**p<0.01. FIGS. 11K and 11M are graphs quantifying calcium imaging inHEK293T cells expressing TRPA1 and TRPV1. The percentage of cells thatrespond to 100 nM MO and 10 μM CA was significantly lower in theTmem100-3Q-transfected group compared with the group lacking Tmem100-3Q(repeated 3 times; *p<0.05, **p<0.01). The statistic is one-way ANOVAwith Bonferroni's post-hoc test; *p<0.05, **p<0.01. All error barsrepresent SEM.

FIG. 12A-FIG. 12K are related to FIG. 4A-4E. FIGS. 12A-12D demonstratescontext-dependent regulation of Tmem100 in the TRPA1-V1 complex. FIGS.12A and 12B are graphs quantifying MO (10 μM)- (FIG. 12A) and CAP (100nM)-induced (FIG. 12B) single-channel activity (NP_(o)) at Vh=−60 mV inCHO cells expressing TRPA1 vs. TRPA1+Tmem100 (1:4 molar ratio) andTRPA1+TRPV1 (1:1) vs. TRPA1+TRPV1+Tmem100 (1:1:4). NP_(o) measurementsaccounted for all recorded conductance, including main andsub-conductance. The configuration is cell-attached patch, and thenumber of patches that responded among those analyzed is indicatedwithin bars. NP_(o): MO 0.6±0.11 for TRPA1+TRPV1 vs. 1.05±0.14 forTRPA1+TRPV1+Tmem100 CHO cells; 1.15±0.15 for TRPA1 vs. 0.64±0.18 forTRPA1+Tmem100 CHO cells. CAP-0.81±0.12 for TRPA1+TRPV1 vs. 0.82±0.14 forTRPA1+TRPV1+Tmem100 CHO cells; 0.81±0.07 for TRPV1 vs. 0.45±0.1 forTRPV1+Tmem100 cells. The statistic is unpaired t-test and one-way ANOVAwith Bonferroni's post-hoc test (*p<0.05; ***p<0.001). All error barsrepresent SEM. FIGS. 12C and 12D show representative single-channelrecording during 5 sec at Vh=−60 mV for MO-gated current (FIG. 12C) and4 sec for CAP-gated current (FIG. 12D) in TRPA1− andTRPA1+Tmem100-expressing CHO cells. Traces are 4 sec long, and theproteins expressed are indicated below the traces. Vertical bars to theright of the traces represent 4 pA; c is the closed state; o1, o2, ando3 show open states for 3 independent channels in the patch. FIGS.12E-12J show characterization of single channel current-voltagerelationships in the presence and absence of Tmem100 and Tmem100-3Qmutant. FIGS. 12E, 12G and 12I are graphs of mustard oil (MO; 10μM)-gated single channel current-voltage relationship (I-V) in CHO cellsexpressing TRPA1, TRPA1+Tmem100 (1:4 molar ratio), TRPA1+TRPV1 (1:1), orTRPA1+TRPV1+Tmem100 (1:1:4) (FIG. 12E); TRPA1 and TRPA1+Tmem100-3Q (1:4)(FIG. 12G); TRPA1+TRPV1 (1:1) and TRPA1+TRPV1+Tmem100-3Q (1:1:4) (FIG.12I). Configuration is cell-attached patch. TRPA1 and TRPV1co-expression in the patches was confirm by responses to both MO andCAP. I-V is presented for main single-channel conductance. Number ofcells recorded: n=5-8. FIGS. 12F, 12J and 12J are graphs of CAP (100nM)-gated single channel I-V relationship in CHO cells expressing TRPV1,TRPV1+Tmem100 (1:4), TRPA1+TRPV1 (1:1) and TRPA1+TRPV1+Tmem100 (1:1:4)(FIG. 12F); TRPV1 and TRPV1+Tmem100-3Q (1:4) (FIG. 12H); TRPA1+TRPV1(1:1), or TRPA1+TRPV1+Tmem100-3Q (1:1:4) (FIG. 12J). The configurationis cell-attached patch. CHO cells co-expressing TRPA1 and TRPV1responded to both CAP and MO. I-V is presented for main single-channelconductance. Number of cells recorded: n=5-7. FIG. 12K is a graphquantifying whole cell recording of capsaicin (100 nM)-evoked currentsin MO (50 μM)-unresponsive (MO−) and -responsive (MO+) DRO neurons fromAvil-Cre;Tmem100^(+/+) and Avil-Cre;Tmem^(fl/fl) mice. The numbers inthe boxes represent the numbers of capsaicin responsive neurons tested.All bars are presented as mean±SEM.

FIG. 13A-FIG. 13D are related to FIG. 5A-FIG. 5F and demonstrate theinteraction among Tmem100, TRPA1, and TRPV1. FIG. 13A is a graphicquantification of GST pull-down for the C-terminus and TRPV1. Increasedassociation between TRPV1 and the C terminus in Tmem100-3Q was observed.N=4, **p<0.01. FIG. 13B is an image of GST pull-down of TRPV1 withdifferent fragments of Tmem100 tagged with GST. TRPV1 pull-down isenhanced in the GST-C-3Q group compared to the GST-C-WT group. FIG. 13Cis a graph of FRET-TIRF measurements of the interaction among TRPV1 andTmem100 (T100). The interaction between Tmem100 and TRPV1 is not alteredby the addition of TRPA1. FIG. 13D is a graph of FRET-TIRF measurementsof the interaction among TRPA1 and Tmem100 (T100). The interactionbetween TRPA1 and Tmem100 is significantly increased with the presenceof TRPV1. Similar to the experiments in FIG. 5A-5F, the Rho-pYC was usedas a positive control for maximal effects of the system, and M-V1 was anegative control. *p<0.05, **p<0.01, ***p<0.001. All bars are presentedas mean±SEM.

FIG. 14A-FIG. 14D are related to FIG. 6A-6G and demonstrate thecontext-dependent regulation of Tmem100-3Q in the TRPA1-V1 complex.FIGS. 14A and 14B are graphs of MO (10 μM)- (FIG. 14A) and CAP (100nM)-induced (FIG. 14B) single-channel activity (NP_(o)) at Vh=−60 mV inCHO cells expressing TRPA1 vs. TRPA1+Tmem100-3Q (T100-3Q) (1:4 molarratio) and TRPA1+TRPV1 (1:1) vs. TRPA1+TRPV1+Tmem100-3Q (1:1:4). NP_(o):MO 0.55±0.09 for TRPA1+TRPV1 vs. 0.16±0.06 for TRPA1+TRPV1+Tmem100-3QCHO cells; 0.98±0.18 for TRPA1 vs. 1.05±0.14 for TRPA1+Tmem100-3Q cells.CAP 1.04±0.23 for TRPA1+TRPV1 vs. 1.08±0.17 for TRPA1+TRPV1+Tmem100-3Qcells; 1.3±0.16 for TRPV1 vs. 0.73±0.12 for TRPV1+Tmem100-3Q cells. Thestatistic is unpaired t-test and one-way ANOVA with Bonferroni'spost-hoc test (*p<0.05; **p<0.01; NS: no significant difference). Allerror bars represent SEM. FIGS. 14 C and D show representativesingle-channel recording traces at Vh=−60 mV for MO-gated current inTRPA1 and TRPA1+Tmem100-3Q-expressing CHO cells (FIG. 14C) and CAP-gatedcurrent in TRPV1 and TRPV1+Tmem100-3Q-expressing CHO cells (FIG. 14D).Traces are 4 sec long, and protein expression is indicated below thetraces. Vertical bars on the right side of the traces represent 4 pA; cis the closed state; o1, o2, and o3 show open states for 3 independentchannels in the patch.

FIG. 15A-FIG. 15D are related to FIG. 7A-7I and demonstrate a shortenedversion of cell-permeable peptide (CPP) derived from T100-Mut, F2, isalso effective for inhibiting TRPA1 activities in the heterologoussystem and painful behavioral models. FIG. 15A is a graph of calciumimaging data from HEK293 cells expressing TRPA1 and TRPV1. Among thethree shortened CPP (sequences on top), only F2 (2 μM) significantlyreduced the percentage of cells responsive to CA (10 μM) compared to thescrambled (repeated 3 times). In FIG. 15A: top sequence (Tmem100): SEQID NO: 8; F1: SEQ ID NO: 16; F2: SEQ ID NO: 17; and F3: SEQ ID NO: 18.*p<0.05. As shown in FIG. 15B, F2 (SEQ ID NO: 17) (5 μl 100 μM;intradermal injection) is also effective in suppressingpaclitaxel-induced mechanical hyperalgesia. N=5, ***p<0.01. FIGS. 15Cand 15D are graphs showing open box: scrambled; black box: F2.Intrathecal injection of human T100-3Q peptide (h-T100) and HC-030031dose-dependently inhibits neuropathic mechanical hypersensitivity inrats. FIG. 15C shows graphs quantifying that in rats with spinal nerveligation (SNL), intrathecal injection of h-T100 (10-100 μM, 10n=7-8/dose) dose-dependently increased ipsilateral paw withdrawalthresholds (PWT) from pre-drug levels, reflecting attenuated mechanicalhypersensitivity. Intrathecal injections of scrambled peptide (100 μM,10 μl, n=6) and vehicle (n=5) had no effect. There was no significantchange in the contralateral PWT after drug injection. FIG. 15D is agraph quantifying the intrathecal injection of HC-030031 (10-200 μM, 10μl, n=6/dose), a selective TRPA1 receptor antagonist, also increasedipsilateral PWTs of SNL rats in a dose-dependent fashion. Intrathecalinjection of vehicle (n=6) was not effective. Contralateral PWTs did notchange significantly after drug treatment. T100-Mut peptide has a lowerEC₅₀ for inhibiting pain (29.3 μM, 10 μl) than HC030031 (58.2 μM, 10μl). *P<0.05, versus pre-drug. One-way repeated measures ANOVA. All barsare presented as mean±SEM.

FIG. 16A-FIG. 16B demonstrate that P2-Mut (T100-Mut; SEQ ID NO: 7)inhibits ongoing pain. FIG. 16A is a graph showing that in rats withspinal nerve ligation (SNL), intrathecal injection of P2-Mut (50 μM, 10μl, n=10) increased the time that rats spent in their drug-pairedchamber, with a corresponding decrease in the scramble peptide-pairedchamber. The difference score (difference score=“Post-conditioningtime”−“Pre-conditioning time”) in the P2-Mut-paired chamber wassignificantly greater than that in scramble-paired chambers, reflectingattenuated ongoing neuropathic pain. However, P2-Mut did not inducechamber preference in sham-operated rats (n=8). This result suggeststhat the drug is not rewarding in the absence of nerve injury, and thepreference to the P2-Mut-paired chamber in SNL rats result fromdrug-induced pain relief. FIG. 16B is a graph showing that, in aseparate experiment, systemic administration of gabapentin (60 mg/kg,i.p.) significantly increased the time the SNL rats spent in thegabapentin-paired chamber (n=8), with a corresponding decrease in thesaline-paired chamber. However, gabapentin did not induce chamberpreference in sham-operated rats (n=8). The difference scores weresignificantly different between gabapentin-paired and saline-pairedchambers in SNL rats, but not in sham-operated rats. *p<0.05 by pairedt-test.

FIG. 17 is a graph that shows deceased itch behavior in Pirt2-CKO(Tmem100-CKO) mice. The Y axis represents the numbers of scratches inthe first 30 minutes after histamine back injection. The Pirt2-CKO(Tmem100 CKO) mice showed robust decreased itch behavior. (n=10 and 11for control and CKO).

FIG. 18 is a graph that shows in the wild-type mice with spinal nerveligation (SNL), mechanical hyperalgesia is observed 6 days postligation. Intradermal injection of Tmem 100 peptide (CPP) SEQ ID NO: 17blocked SNL-induced mechanical hyperalgesia.

DETAILED DESCRIPTION OF THE INVENTION

TRPA1 and TRPV1 are crucial mediators of pain and have been shown toform Complexes. The present invention is based, at least in part, on theidentification of Tmem100 peptides and mutants or derivatives thereof,as a potentiating modulator of TRPA1-V1 complexes.

Pain is the cardinal symptom of many debilitating diseases, causingheavy societal and health burdens worldwide. It is known that ionchannels and receptors in the dorsal root ganglia (DRG) are responsiblefor the detection of noxious stimuli, and their plasticity contributesto the increased severity of pain (Woolf and Costigan, 1999). TRP(Transient Receptor Potential) channels are emerging targets forunderstanding this process and developing treatments (Venkatachalam andMontell, 2007). Their ability to form multimeric complexes (Goel et al.,2002; Hellwig et al., 2005; Hofmann et al., 2002; Schaefer, 2005;Strübing et al., 2001; Xu et al., 1997) broadens the variety andcomplexity of channel regulation and the potential implications for painmodulation (Jeske et al., 2011; Liu et al., 2011; Patil et al., 2011;Schmidt et al., 2009). Among TRP channels, TRPA1 and TRPV1 are essentialand widely studied molecular sensors and mediators of pain signals inDRG neurons (Bautista et al., 2006; Caterina et al., 2000; Caterina etal., 1997). It is well documented that most if not all TRPA1⁺ DRGneurons co-express TRPV1 (Bautista et al., 2006; Story et al., 2003).Although recent studies have suggested that TRPA1 and TRPV1 can form acomplex in a heterologous expression system as well as sensory neurons(Fischer et al., 2014; McMahon and Wood, 2006; Salas et al., 2009;Staruschenko et al., 2010), the functional significance and modulationof the complex in the nociceptive pathway are unclear.

Tmem100 (also known as Pirt2) was identified as a candidate for themodulation of the TRPA1-V1 complex in the nociceptive pathway. Tmem100is a 134-amino acid, two-transmembrane protein highly conserved invertebrates (Moon et al., 2010). It is found in other organs besides theDRG, expressed in blood vessels, ventral neural tubes, and the notochord(Moon et al., 2010). Tmem100 has been shown to be involved in processessuch as renal development (Georgas et al., 2009), vasculogenesis (Moonet al., 2010), and lung cancer cell invasiveness (Frullanti et al.,2012). However, prior to the invention described herein, little wasknown about the underlying mechanisms of these effects and the role ofTmem100 in the nervous system.

Data described herein demonstrate that Tmem100 enhances TRPA1 activityin vitro and in vivo. Interestingly, this regulation depends on thepresence of TRPV1. In the DRG, Tmem100 is co-expressed with TRPA1 andTRPV1. It forms a complex with TRPA1 and TRPV1 in both DRG neurons andheterologous systems. Tmem100 selectively augments TRPA1-associatedactivity by increasing the open probability of the channel when TRPA1and TRPV1 are both present in membrane patches. Tmem100 mutant miceexhibit a reduction in inflammatory mechanical hyperalgesia andTRPA1-but not TRPV1-mediated pain. Mechanistically, Tmem100 weakens theassociation of TRPA1 and TRPV1, thereby releasing the inhibition ofTRPA1 by TRPV1 (Salas et al., 2009). A Tmem100 mutant, Tmem100-3Q,exerts the opposite effect, i.e., it enhances the association of TRPA1and TRPV1 and strongly inhibits TRPA1. Taking advantage of thisinhibition, a new strategy was developed and described herein forblocking persistent pain. A cell-permeable peptide that mimics theC-terminus of Tmem100-3Q and selectively inhibits TRPA1-mediatedactivity and pain in a TRPV1-dependent manner is described herein.

TRP channels have been classified into at least six groups: TRPC(short), TRPV (vanilloid), TRPM (long, melastatin), TRPP (polycystins),TRPML (mucolipins), and TRPA (ANKTM1). The TRPC group can be dividedinto 4 subfamilies (TRPC1, TRPC4,5, TRPC3,6,7 and TRPC2) based onsequence homology and functional similarities. Currently the TRPV familyhas 6 members. TRP V5 and TRP V6 are more closely related to each otherthan to TRPV1, TRP V2, TRPV3, or TRPV4. TRPA1 is most closely related toTRPV3, and is more closely related to TRPV1 and TRPV2 than to TRPV5 andTRPV6. The TRPM family has 8 members. Constituents include thefollowing: the founding member TRPM1 (Melastatin or LTRPC1), TRPM3(KIAA1 616 or LTRPC3), TRPM7 (TRP-PLIK, ChaK(1), LTRPC7), TRPM6 (ChaK2),TRPM2 (TRPC7 or LTRPC2), TRPM8 (Trp-p8 or CMR1), TRPM5 (Mtr1 or LTRPC5),and TRPM4 (FLJ20041 or LTRPC4). The sole mammalian member of the TRPAfamily is ANKTM1. The TRPML family consists of the mucolipins, whichinclude TRPML1 (mucolipins 1), TRPML2 (mucolipins 2), and TRPML3(mucolipin3). The TRPP family consists of two groups of channels: thosepredicted to have six transmembrane domains and those that have 11.TRPP2 (PKD2), TRPP3 (PKD2L1), TRPP5 (PKD2L2) are all predicted to havesix transmembrane domains. TRPP1 (PKD13 PCl)5 PKD-REJ and PKD-IL1 areall thought to have 11 transmembrane domains.

The TRP channels constitute a large and important class of channelsinvolved in modulating cellular homeostasis. The present inventionprovides methods and agents that modulate at least one TRP familymember. Specifically, the present invention provides methods and agentsfor antagonizing a function of TRPA1. Modulating a function of TRPA1provides a means for modulating calcium homeostasis, sodium homeostasis,intracellular calcium levels, membrane polarization (resting membranepotential), and/or cation levels in a cell. Agents that can modulate oneor more TRPA1 functions are useful in many aspects including, but notlimited to, maintaining calcium homeostasis; maintaining sodiumhomeostasis; modulating intracellular calcium levels; modulatingmembrane polarization (membrane potential); modulating cation levels;and/or treating or preventing diseases, disorders, or conditionsassociated with calcium homeostasis, sodium homeostasis, calcium orsodium dyshomeostasis, or membrane polarization/hyperpolarization(including hypo and hyperexcitability), and/or treating or preventingdiseases, disorders, or conditions associated with regulation ormisregulation of TRPA1 expression or function.

The present application provides agents that can modulate TRPA1 functionas well as methods employing said agents.

Preferably, the Tmem 100 mutant polypeptides described herein comprise alipid modification, e.g., palmitoyl (Pal) or myristyl (Myr) groups, attheir N-terminus to make them cell permeable.

Palmitoylation is the covalent attachment of fatty acids, such aspalmitic acid, to cysteine, serine, or threonine residues of proteins,which are typically membrane proteins. Palmitoylation enhances thehydrophobicity of proteins and contributes to their membraneassociation. Palmitoylation also plays a significant role in subcellulartrafficking of proteins between membrane compartments, as well as inmodulating protein-protein interactions. In contrast to prenylation andmyristoylation, palmitoylation is usually reversible (because the bondbetween palmitic acid and protein is often a thioester bond). Thereverse reaction is catalysed by palmitoyl protein thioesterases.Because palmitoylation is a dynamic, post-translational process, it isemployed by the cell to alter the subcellular localization,protein-protein interactions, or binding capacities of a protein.

Myristoylation is an irreversible, protein lipidation modification wherea myristoyl group, derived from myristic acid, is covalently attached byan amide bond to the alpha-amino group of an N-terminal glycine residue.Myristic acid is a 14-carbon saturated fatty acid (14:0) with thesystematic name of n-Tetradecanoic acid. This modification can be addedeither co-translationally or post-translationally.N-myristoyltransferase (NMT) catalyzes the myristic acid additionreaction in the cytoplasm of cells. This lipidation event is commonamong many organisms including animals, plants, fungi, protozoans andviruses. Myristoylation allows for weak protein-protein andprotein-lipid interactions and plays an essential role in membranetargeting, protein-protein interactions and functions widely in avariety of signal transduction pathways.

Compositions or Agents

In one aspect, the present invention provides compositions or agents andpharmaceutical compositions comprising, consisting of, or consistingessentially of particular TRPA1 inhibitory polypeptides and nucleicacids.

As used herein, the term “isolated” when used to refer to nucleic acidand polypeptide compositions refers to nucleic acids or polypeptidesexisting in a state other than the state in which they exist in nature.In other words, the term is used to denote some level of separation fromother proteins and cellular components with which the protein isendogenously found. Isolated, when used in this context, does notnecessarily mean that the protein or nucleic acid is provided in apurified form. Additionally, the term “isolated” is not intended toimply that the polypeptide or nucleic acid is isolated from an organism.Rather, the term also includes recombinantly produced nucleic acids andpolypeptides.

The present invention also encompasses isolated polynucleotides thatencode a polypeptide comprising Tmem100 peptide or fragment thereof orTmem100 mutant peptide or fragment thereof. The present invention alsoencompasses he isolated polypeptides.

The term “polynucleotide encoding a polypeptide” encompasses apolynucleotide which includes only coding sequences for the polypeptideas well as a polynucleotide which includes additional coding and/ornon-coding sequences. The polynucleotides of the invention can be in theform of RNA or in the form of DNA. DNA includes cDNA, genomic DNA, andsynthetic DNA; and can be double-stranded or single-stranded, and ifsingle stranded can be the coding strand or non-coding (anti-sense)strand.

The invention provides an isolated polypeptide comprising, consistingof, or consisting essentially of the amino acid sequence represented inSEQ ID NO: 5 or SEQ ID NO: 6, or fragments thereof.

The invention provides an isolated polypeptide comprising, consistingof, or consisting essentially of the amino acid sequence represented inSEQ ID NO: 7 or SEQ ID NO: 8, or fragments thereof.

The invention provides an isolated polypeptide encoded by a nucleic acidsequence comprising, consisting of, or consisting essentially of SEQ IDNO: 9 or SEQ ID NO: 10.

The invention provides an isolated polypeptide encoded by a nucleic acidsequence comprising, consisting of, or consisting essentially of anucleotide sequence represented in SEQ ID NO: 11 or SEQ ID NO: 12.

The present invention further relates to variants of thepolynucleotides, for example, fragments, analogs, and derivatives. Thevariant of the polynucleotide can be a naturally occurring allelicvariant of the polynucleotide or a non-naturally occurring variant ofthe polynucleotide. The polynucleotide can have a coding sequence whichis a naturally occurring allelic variant of the coding sequence of thedisclosed polypeptides. As known in the art, an allelic variant is analternate form of a polynucleotide sequence that have, for example, asubstitution, deletion, or addition of one or more nucleotides, whichdoes not substantially alter the function of the encoded polypeptide.

The polynucleotides can comprise the coding sequence for the maturepolypeptide fused in the same reading frame to a polynucleotide whichaids, for example, in expression and secretion of a polypeptide from ahost cell (e.g., a leader sequence which functions as a secretorysequence for controlling transport of a polypeptide from the cell). Thepolypeptide having a leader sequence is a preprotein and can have theleader sequence cleaved by the host cell to form the mature form of thepolypeptide. The polynucleotides can also encode for a proprotein whichis the mature protein plus additional 5′ amino acid residues. A matureprotein having a prosequence is a proprotein and is an inactive form ofthe protein. Once the prosequence is cleaved an active mature proteinremains.

The polynucleotides can comprise the coding sequence for the maturepolypeptide fused in the same reading frame to a marker sequence thatallows, for example, for purification of the encoded polypeptide. Forexample, the marker sequence can be a hexa-histidine tag supplied by apQE-9 vector to provide for purification of the mature polypeptide fusedto the marker in the case of a bacterial host, or the marker sequencecan be a hemagglutinin (HA) tag derived from the influenza hemagglutininprotein when a mammalian host (e.g., COS-7 cells) is used. Additionaltags include, but are not limited to, Calmodulin tags, FLAG tags, Myctags, S tags, SBP tags, Softag 1, Softag 3, V5 tag, Xpress tag,Isopeptag, SpyTag, Biotin Carboxyl Carrier Protein (BCCP) tags, GSTtags, fluorescent protein tags (e.g., green fluorescent protein tags),maltose binding protein tags, Nus tags, Strep-tag, thioredoxin tag, TCtag, Ty tag, and the like.

The present invention provides isolated nucleic acid molecules having anucleotide sequence at least 80% identical, at least 85% identical, atleast 90% identical, at least 95% identical, or at least 96%, 97%, 98%or 99% identical to a polynucleotide comprising, consisting of, orconsisting essentially of the amino acid sequence represented in SEQ IDNO: 1 or SEQ ID NO: 3, or fragments thereof.

By a polynucleotide having a nucleotide sequence at least, for example,95% “identical” to a reference nucleotide sequence is intended that thenucleotide sequence of the polynucleotide is identical to the referencesequence except that the polynucleotide sequence can include up to fivepoint mutations per each 100 nucleotides of the reference nucleotidesequence. In other words, to obtain a polynucleotide having a nucleotidesequence at least 95% identical to a reference nucleotide sequence, upto 5% of the nucleotides in the reference sequence can be deleted orsubstituted with another nucleotide, or a number of nucleotides up to 5%of the total nucleotides in the reference sequence can be inserted intothe reference sequence. These mutations of the reference sequence canoccur at the amino- or carboxy-terminal positions of the referencenucleotide sequence or anywhere between those terminal positions,interspersed either individually among nucleotides in the referencesequence or in one or more contiguous groups within the referencesequence.

As a practical matter, whether any particular nucleic acid molecule isat least 80% identical, at least 85% identical, at least 90% identical,and in some embodiments, at least 95%, 96%, 97%, 98%, or 99% identicalto a reference sequence can be determined conventionally using knowncomputer programs such as the Bestfit program (Wisconsin SequenceAnalysis Package, Version 8 for Unix, Genetics Computer Group,University Research Park, 575 Science Drive, Madison, Wis. 53711).Bestfit uses the local homology algorithm of Smith and Waterman,Advances in Applied Mathematics 2: 482 489 (1981), to find the bestsegment of homology between two sequences. When using Bestfit or anyother sequence alignment program to determine whether a particularsequence is, for instance, 95% identical to a reference sequenceaccording to the present invention, the parameters are set such that thepercentage of identity is calculated over the full length of thereference nucleotide sequence and that gaps in homology of up to 5% ofthe total number of nucleotides in the reference sequence are allowed.

The polynucleotide variants can contain alterations in the codingregions, non-coding regions, or both. In some examples, thepolynucleotide variants contain alterations which produce silentsubstitutions, additions, or deletions, but do not alter the propertiesor activities of the encoded polypeptide. In some examples, nucleotidevariants are produced by silent substitutions due to the degeneracy ofthe genetic code. Polynucleotide variants can be produced for a varietyof reasons, e.g., to optimize codon expression for a particular host(change codons in the human mRNA to those preferred by a bacterial hostsuch as E. coli).

The terms “identical” or “percent identity” in the context of two ormore nucleic acids or polypeptides, refer to two or more sequences orsubsequences that are the same or have a specified percentage ofnucleotides or amino acid residues that are the same, when compared andaligned (introducing gaps, if necessary) for maximum correspondence, notconsidering any conservative amino acid substitutions as part of thesequence identity. The percent identity is be measured using sequencecomparison software or algorithms or by visual inspection. Variousalgorithms and software are known in the art that is used to obtainalignments of amino acid or nucleotide sequences. One such non-limitingexample of a sequence alignment algorithm is the algorithm described inKarlin et al., Proc. Natl. Acad. Sci., 87:2264-2268 (1990), as modifiedin Karlin et al., Proc. Natl. Acad. Sci., 90:5873-5877 (1993), andincorporated into the NBLAST and XBLAST programs (Altschul et al.,Nucleic Acids Res., 25:3389-3402 (1991)). Gapped BLAST is used asdescribed in Altschul et al., Nucleic Acids Res. 25:3389-3402 (1997).BLAST-2, WU-BLAST-2 (Altschul et al., Methods in Enzymology, 266:460-480(1996)), ALIGN, ALIGN-2 (Genentech, South San Francisco, Calif.) orMegalign (DNASTAR) are additional publicly available software programsthat can be used to align sequences. The percent identity between twonucleotide sequences is determined using the GAP program in GCG software(e.g., using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70,or 90 and a length weight of 1, 2, 3, 4, 5, or 6). The GAP program inthe GCG software package, which incorporates the algorithm of Needlemanand Wunsch (J. Mol. Biol. (48):444-453 (1970)) is used to determine thepercent identity between two amino acid sequences (e.g., using either aBlossum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12,10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5). The percentidentity between nucleotide or amino acid sequences is determined usingthe algorithm of Myers and Miller (CABIOS, 4:11-17 (1989)). For example,the percent identity is determined using the ALIGN program (version 2.0)and using a PAM120 with residue table, a gap length penalty of 12 and agap penalty of 4. Appropriate parameters for maximal alignment byparticular alignment software can be determined by one skilled in theart. In some examples, the default parameters of the alignment softwareare used. In some examples, the percentage identity “X” of a first aminoacid sequence to a second sequence amino acid is calculated as100×(Y/Z), where Y is the number of amino acid residues scored asidentical matches in the alignment of the first and second sequences (asaligned by visual inspection or a particular sequence alignment program)and Z is the total number of residues in the second sequence. If thelength of a first sequence is longer than the second sequence, thepercent identity of the first sequence to the second sequence will belonger than the percent identity of the second sequence to the firstsequence.

As a non-limiting example, whether any particular polynucleotide has acertain percentage sequence identity (e.g., is at least 80% identical,at least 85% identical, at least 90% identical, and in some examples, atleast 95%, 96%, 97%, 98%, or 99% identical) to a reference sequence can,in certain examples, he determined using the Bestfit program (WisconsinSequence Analysis Package, Version 8 for Unix, Genetics Computer Group,University Research Park, 575 Science Drive, Madison, Wis. 53711).Bestfit uses the local homology algorithm of Smith and Waterman,Advances in Applied Mathematics 2: 482 489 (1981), to find the bestsegment of homology between two sequences. When using Bestfit or anyother sequence alignment program to determine whether a particularsequence is, for instance, 95% identical to a reference sequenceaccording to the present invention, the parameters are set such that thepercentage of identity is calculated over the full length of thereference nucleotide sequence and that gaps in homology of up to 5% ofthe total number of nucleotides in the reference sequence are allowed.

In an example, two nucleic acids or polypeptides of the invention aresubstantially identical, meaning they have at least 70%, at least 75%,at least 80%, at least 85%, at least 90%, and in some examples at least95%, 96%, 97%, 98%, 99% nucleotide or amino acid residue identity, whencompared and aligned for maximum correspondence, as measured using asequence comparison algorithm or by visual inspection. Identity canexist over a region of the sequences that is at least about 5, at leastabout 10, about 20, about 40-60 residues in length or any integral valuetherebetween, or over a longer region than 60-80 residues, at leastabout 90-100 residues, or the sequences are substantially identical overthe full length of the sequences being compared.

A “conservative amino acid substitution” is one in which one amino acidresidue is replaced with another amino acid residue having a similarside chain. Families of amino acid residues having similar side chainshave been defined in the art, including basic side chains (e.g., lysine,arginine, histidine), acidic side chains (e.g., aspartic acid, glutamicacid), uncharged polar side chains (e.g., glycine, asparagine,glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains(e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine,methionine, tryptophan), beta-branched side chains (e.g., threonine,valine, isoleucine) and aromatic side chains (e.g., tyrosine,phenylalanine, tryptophan, histidine). For example, substitution of aphenylalanine for a tyrosine is a conservative substitution. Preferably,conservative substitutions in the sequences of the polypeptides andantibodies of the invention do not abrogate the binding of thepolypeptide or antibody containing the amino acid sequence, to theantigen(s). Methods of identifying nucleotide and amino acidconservative substitutions which do not eliminate antigen binding arewell-known in the art (see, e.g., Brummell et al., Biochem. 32: 1180-1187 (1993); Kobayashi et al. Protein Eng. 12(10):879-884 (1999); andBurks et al. Proc. Natl. Acad. Sci. USA 94:412-417 (1997)).

The polypeptides of the present invention can be recombinantpolypeptides, natural polypeptides, or synthetic polypeptides. It willbe recognized in the art that some amino acid sequences of the inventioncan be varied without significant effect of the structure or function ofthe protein. Such mutants include deletions, insertions, inversions,repeats, and type substitutions.

The polypeptides and analogs can be further modified to containadditional chemical moieties not normally part of the protein. Thosederivatized moieties can improve the solubility, the biologicalhalf-life or absorption of the protein. The moieties can also reduce oreliminate any desirable side effects of the proteins and the like. Anoverview for those moieties can be found in Remington's PharmaceuticalSciences, 20th ed., Mack Publishing Co., Easton, Pa. (2000).

The isolated polypeptides described herein can be produced by anysuitable method known in the art. Such methods range from direct proteinsynthetic methods to constructing a DNA sequence encoding isolatedpolypeptide sequences and expressing those sequences in a suitabletransformed host. In some examples, a DNA sequence is constructed usingrecombinant technology by isolating or synthesizing a DNA sequenceencoding a wild-type protein of interest. Optionally, the sequence canbe mutagenized by site-specific mutagenesis to provide functionalanalogs thereof. See, e.g. Zoeller et al., Proc. Nat'l. Acad. Sci. USA81:5662-5066 (1984) and U.S. Pat. No. 4,588,585.

A DNA sequence encoding a polypeptide of interest could be constructedby chemical synthesis using an oligonucleotide synthesizer. Sucholigonucleotides can be designed based on the amino acid sequence of thedesired polypeptide and selecting those codons that are favored in thehost cell in which the recombinant polypeptide of interest will beproduced. Standard methods can be applied to synthesize an isolatedpolynucleotide sequence encoding an isolated polypeptide of interest.For example, a complete amino acid sequence can be used to construct aback-translated gene. Further, a DNA oligomer containing a nucleotidesequence coding for the particular isolated polypeptide can besynthesized. For example, several small oligonucleotides coding forportions of the desired polypeptide can be synthesized and then ligated.The individual oligonucleotides typically contain 5′ or 3′ overhangs forcomplementary assembly.

The invention provides an expression vector, which replicates in atleast one of a prokaryotic cell and eukaryotic cell. The expressionvector comprises any of the foregoing TRPA1 inhibitory nucleic acids.Similarly provided are cells comprising these expression vectors, whichcells express the TRPA1 inhibitory protein encoded by the expressednucleic acid.

Additionally provided are methods of producing a polypeptide. The methodincludes culturing one of the foregoing cells (e.g., a cell expressing aTRPA1 inhibitory polypeptide) in a suitable cell culture medium toexpress said polypeptide. As noted above, the invention contemplates anexpression vector which comprises a coding sequence for a TRPA1inhibitory protein, as provided herein. A “vector” is a replicon, suchas plasmid, phage or cosmid, to which another DNA segment is attached.The term “vector” refers to a nucleic acid molecule capable oftransporting another nucleic acid to which it has been linked. One typeof vector is an episome which is a nucleic acid capable ofextra-chromosomal replication. Vectors capable of autonomous replicationand/or expression of nucleic acids to which they are linked is alsoused. Vectors capable of directing the expression of genes to which theyare operatively linked are referred to herein as “expression vectors.”In general, expression vectors of utility in recombinant DNA techniquesare often in the form of “plasmids” which refer generally to circulardouble stranded DNA loops which, in their vector faun are not bound tothe chromosome. However, the invention is intended to include suchother, forms of expression vectors which serve equivalent functions andwhich become known in the art subsequently hereto.

A DNA or nucleic acid “coding sequence” is a DNA sequence which istranscribed and translated into a polypeptide in vivo when placed underthe control of appropriate regulatory sequences. The boundaries of thecoding sequence are determined by a start codon at the 5′ (amino)terminus and a translation stop codon at the 3′ (carboxyl) terminus. Acoding sequence of the present invention can include, but is not limitedto, cDNA from eukaryotic mRNA, genomic DNA sequences from eukaryotic(e.g., mammalian) DNA, and synthetic DNA sequences. A polyadenylationsignal and transcription termination sequence is located 3′ of thecoding sequence.

Nucleic acid or DNA regulatory sequences or regulatory elements aretranscriptional and translational control sequences, such as promoters,enhancers, polyadenylation signals, and terminators, that provide forand/or regulate expression of a coding sequence in a host cell.Regulatory sequences for directing expression of eukaryotic ion channelsand detectable markers of certain examples are art-recognized and isselected by a number of well understood criteria. Examples of regulatorysequences are described in Goeddel, Gene Expression Technology: Methodsin Enzymology (Academic Press, San Diego, Calif. (1990)). For instance,any of a wide variety of expression control sequences that control theexpression of a DNA sequence when operatively linked to it is used inthese vectors to express DNA sequences encoding the ion channels anddetectable markers. Such useful expression control sequences, include,for example, the early and late promoters of SV40, beta2 tubulin,adenovirus or cytomegalovirus immediate early promoter, the lac system,the trp system, the TAC or TRC system, T7 promoter whose expression isdirected by T7 RNA polymerase, the promoter for 3-phosphoglyceratekinase or other glycolytic enzymes, the promoters of acid phosphatase,e.g., Pho5₅ and the promoters of the yeast α-mating factors and othersequences known to control the expression of genes of prokaryotic oreukaryotic cells or their viruses, and various combinations thereof. Itshould be understood that the design of the expression vector depends onsuch factors as the choice of the host cell to be transformed. Moreover,the vector's copy number, the ability to control that copy number andthe expression of any other protein encoded by the vector, such asantibiotic markers, should also be considered.

The invention contemplates the use of any promoter that can drive theexpression of a TRPA1 inhibitory protein in prokaryotic or eukaryoticcells. As used herein, the term “promoter” means a DNA sequence thatregulates expression of a selected DNA sequence operably linked to thepromoter, and which effects expression of the selected DNA sequence incells. A “promoter” generally is a DNA regulatory element capable ofbinding RNA polymerase in a cell and initiating transcription of acoding sequence. For example, the promoter sequence is bounded at its 3′terminus by the transcription initiation site and extend upstream (5′direction) to include the minimum number of bases or elements necessaryto initiate transcription at levels detectable above background. Withinthe promoter sequence is found a transcription initiation site, as wellas protein-binding domains responsible for the binding of RNApolymerase. Eukaryotic promoters will often, but not always, contain“TATA” boxes and “CANT” boxes. Various promoters, including induciblepromoters, is used to drive the various vectors of the presentinvention. The term “promoter” also encompasses prokaryotic and/oreukaryotic promoters and promoter elements. The term “promoter” as usedherein encompasses “cell specific” promoters, i.e. promoters, whicheffect expression of the selected DNA sequence only in specific cells(e.g., cells of a specific tissue). The term also covers so-called“leaky” promoters, which regulate expression of a selected DNA primarilyin one tissue, but cause expression in other tissues as well. The termalso encompasses non-tissue specific promoters and promoters thatconstitutively express or that, are inducible (i.e., expression levelscan be controlled).

Cells expressing an expression vector are assayed to confirm expressionof TRPA1 inhibitory protein. For example, protein expression isconfirmed using Western blot analysis, immunocytochemistry, orimmunohistochemistry. Additionally or alternatively, TRPA1 function canbe assessed using, for example, calcium imaging analysis to evaluate ionflux or electrophysiological methods (e.g., patch clamp analysis) toevaluate current.

Methods

The present invention provides, generally, methods for treating pain oritch.

In certain aspects, the invention features methods for treating orpreventing a condition associated with TRPA1 function or for whichreduced TRPA1 activity can reduce the severity, comprising administeringan effective amount of a Tmem100 mutant polypeptide, or fragmentthereof.

The present invention also provides methods of preventing, treating, oralleviating symptoms of a disease or condition associated with TRPA1function or for which reduced TRPA1 activity can reduce the severity,comprising administering to a subject in need thereof a Tmem100 mutantpolypeptide, or fragment thereof.

The present invention also provides methods of inhibiting TRPA1 functionin a cell, comprising administering to the cell an effective amount of aTmem100 mutant polypeptide, or fragment thereof, thereby inhibitingTRPA1 function in the cell.

In certain embodiment, the TRPA1 function is an association with TRPV1.

Preferably, the Tmem100 mutant polypeptide, or fragment thereof,enhances the association of TRPA1 with TRPV1.

The TRPA1 function is an inward TRPA1-mediated current, an outwardTRPA1-mediated current or TRPA1-mediated ion flux.

As discussed herein, the Tmem100 mutant polypeptide comprises SEQ ID NO:1, or fragments thereof or SEQ ID NO: 3, or fragments thereof.

In other examples of the methods, the Tmem100 mutant polypeptide, orfragment thereof, is provided to a cell and the cell is a sensoryneuron. The sensory neuron preferably resides in the dorsal root ganglia(DRG).

Any of the methods described herein are used to prevent, treat, oralleviate symptoms of pain or to prevent, treat, or alleviate symptomsof itch.

The Tmem100 mutant polypeptide, or fragment thereof are administeredalone or in combination with one or more agents, as described in moredetail below. The Tmem100 mutant polypeptide, or fragment thereof, isadministered in combination with one or more of a TRPA1 inhibitor, aTRPV3 inhibitor, a TRPV4 inhibitor, or a TRPM8 inhibitor.

In addition to TRPV family members, other TRP channels have beenimplicated in pain reception and/or sensation. For example, certain TRPMchannels including TRPM8 have been implicated in the reception and/orsensation of pain. Accordingly, in certain examples the methods of thepresent invention include treating pain by administering (i) acombination of a TRPA1 inhibitor as described herein and a TRPM8inhibitor; (ii) a combination of a TRPA1 inhibitor, a TRPM8 inhibitor,and one or more of a selective TRPV1 and/or TRPV3 inhibitor; (iii) across-TRP inhibitor that inhibits a function of TRPA1 and TRPM8; or (iv)a pan inhibitor that inhibits a function of TRPA1, TRPM8, and one ormore of TRPV1 and TRPV3.

Diseases and Disorders

The invention provides methods and compositions for inhibiting afunction of a TRPA1 channel in vitro or in vivo. Exemplary functionsinclude, but are not limited to, TRPA1-mediated current. The inventionprovides methods and compositions for inhibiting TRPA1-mediated neuronalhyperexcitability. The invention provides methods for preventing ortreating a disease or disorder or condition by administering acomposition that modulates the level and/or activity of a TRPA1 protein.The composition selectively inhibits the expression level and/oractivity of a TRPA1 protein. In other words, in certain embodiment, thecomposition inhibits the activity of a TRPA1 protein preferentially incomparison to the activity of one or more other ion channels.

In particular examples of the methods for preventing or treatingdiseases and disorders provided herein, the disease or disorder can be,for example, a pain or sensitivity to touch. For example, the pain isrelated to a disease or disorder, e.g., cancer pain, a dermatologicaldisease or disorder, a neurodegenerative disease or disorder, (e.g.,Alzheimer's disease (AD), Parkinson's disease, Huntingdon's disease,amyotrophic lateral sclerosis (ALS)), and other brain disorders causedby trauma or other insults including aging, an inflammatory disease(e.g., asthma, chronic obstructive pulmonary disease, rheumatoidarthritis, osteoarthritis, inflammatory bowel disease,glomerulonephritis, neuroinflammatory diseases, multiple sclerosis, anddisorders of the immune system), cancer (e.g. liposarcoma) or otherproliferative disease, kidney disease and liver disease, a metabolicdisorder such as diabetes. Further diseases and conditions includepost-surgical pain, post herpetic neuralgia, incontinence, and shingles.

Compositions and methods provided herein are used in connection withtreatment of malignancies, including, but not limited to, malignanciesof lymphoreticular origin, bladder cancer, breast cancer, colon cancer,endometrial cancer, head and neck cancer, lung cancer, melanoma, ovariancancer, prostate cancer and rectal cancer, in addition to skin cancersdescribed above. Intracellular calcium level plays an important role incell proliferation in cancer cells (Weiss et al. (2001) InternationalJournal of Cancer 92 (6):877-882).

In addition, pain associated with cancer or with cancer treatment is asignificant cause of chronic pain. Cancers of the bone, for example,osteosarcoma, are considered exceptionally painful, and patients withadvanced bone cancer requires sedation to tolerate the intense andpersistent pain. Accordingly, TRPA1 antagonists of the inventionrepresent a significant possible therapeutic for the treatment of pain,for example, the pain associated with cancer or with cancer treatment.

Given that TRPA1 is differentially expressed in transformed cells, TRPA1blockers also affect the proliferation of transformed cells and thus bea useful way to slow the disease (see Jaquemar et al. (1999) JBC274(11): 7325-33). Thus TRPA1 antagonists could alleviate both the causeand symptoms of cancer pain.

Cancer treatments are not only painful, but they can even be toxic tohealthy tissue. Some chemotherapeutic agents can cause painfulneuropathy. Accordingly, TRPA1 antagonists of the invention represent asignificant possible therapeutic for the treatment of the pain and/orinflammation associated with cancer treatments that cause neuropathy.

In other particular examples of the methods for preventing or treatingdiseases and disorders provided herein, the disease or disorder can bean itch. Many dermatological disorders are accompanied by itch(pruritus). Pruritus and pain share many mechanistic similarities. Bothare associated with activation of C-fibers, both are potentiated byincreases in temperature and inflammatory mediators and both can bequelled with opiates. Decreasing neuronal excitability, particularlyC-fiber excitability alleviates pruritus associated with dialysis,dermatitis, pregnancy, poison ivy, allergy, dry skin, chemotherapy andeczema in some examples.

Compositions and methods provided herein are also used in connectionwith treatment of inflammatory diseases. These diseases include but arenot limited to asthma, chronic obstructive pulmonary disease, rheumatoidarthritis, osteoarthritis, inflammatory bowel disease,glomerulonephritis, neuroinflammatory diseases such as multiplesclerosis, and disorders of the immune’ system.

The compositions that inhibit TRPA1 described herein can be used in thetreatment of any of the foregoing or following diseases or conditions,including in the treatment of pain associated with any of the foregoingor following diseases or conditions. When used in a method of treatment,an inhibitor can be selected and formulated based on the intended routeof administration. Inhibitors can be used to treat the underlyingdisease or condition, or to relieve a symptom of the disease orcondition. Exemplary symptoms include pain associated with a disease orcondition, e.g. sensitivity to pain and touch, or pain-related diseasesor disorders. Compositions and methods provided herein are used inconnection with prevention or treatment of pain or sensitivity to painand touch. Pain or sensitivity to pain and touch are indicated in avariety of diseases, disorders or conditions, including, but not limitedto, diabetic neuropathy, breast pain, psoriasis, eczema, dermatitis,burn, post-herpetic neuralgia (shingles), nociceptive pain, peripheralneuropathic and central neuropathic pain, chronic pain, cancer and tumorpain, spinal cord injury, crush injury and trauma induced pain,migraine, cerebrovascular and vascular pain, sickle cell disease pain,rheumatoid arthritis pain, musculoskeletal pain including treating signsand symptoms of osteoarthritis and rheumatoid arthritis, orofacial andfacial pain, including dental, temporomandibular disorder, and cancerrelated, lower back or pelvic pain, surgical incision related pain,inflammatory and non-inflammatory pain, visceral pain, psychogenic painand soft tissue inflammatory pain, fibromyalgia-related pain, and reflexsympathetic dystrophy, and pain resulting from kidney stones or urinarytract infection.

The compositions and methods of the invention are used in the treatmentof chronic, as well as acute pain. In some examples, chronic or acutepain is the result of injury, age, or disease.

Pain can be generally categorized as chronic pain and acute pain. Thetwo categories of pain differ in duration, as well as underlyingmechanism. Chronic pain is not only persistent, but also does notgenerally respond well to treatment with currently available analgesics,non-steroidal anti-inflammatory drugs, and opioids.

Two broad sub-categories of chronic pain are neuropathic pain and cancerpain. Wang and Wang (2003) Advanced Drug Delivery Reviews 55: 949-965.Neuropathic pain refers to pain resulting from damage (e.g., fromdisease, injury, age) to the nervous system (e.g., nerves, spinal cord,CNS, PNS). In some examples, cancer-related pain is caused by tumorinfiltration, nerve compression, substances secreted by tumors, or theparticular treatment regimen (e.g., radiation, chemotherapeutics,surgery).

Pain is also often classified mechanistically as nociceptive,inflammatory, or neuropathic. Nociceptive pain is pain experiencedfollowing, for example, changes or extremes in temperature, exposure toacids, exposure to chemical agents, exposure to force, and exposure topressure. Reception of painful stimuli sends impulses to the dorsal rootganglia. The response is typically a combination of a reflexive response(e.g., withdrawal from the stimuli) and an emotional reaction.Inflammation is the immune system's response to injury or disease. Inresponse to injury or disease, macrophages, mast cells, neutrophils, andother cells of the immune system are recruited. This infiltration ofcells, along with the release of cytokines and other factors (e.g.,histamine, serotonin, bradykinin, prostaglandins, ATP, H+, nerve growthfactor, TNFα, endothelins, interleukins), can cause fever, swelling, andpain. Current treatments for the pain of inflammation include Cox2inhibitors and opioids. Neuropathic pain refers to pain resulting fromdamage (e.g., from disease, injury, age) to the nervous system- (e.g.,nerves, spinal cord, CNS, PNS). Current treatment for neuropathic painincludes tricyclic antidepressants, anticonvulsants, Na+ channelblockers, NMDA receptor antagonists, and opioids. There are numerousanimal models for studying pain. The various models use various-agentsor procedures to simulate pain resulting from injuries, diseases, orother conditions. Blackburn-Munro (2004) Trends in PharmacologicalSciences 25: 299-305 (see, for example, Table 1). Behavioralcharacteristics of challenged animals can then be observed. Compounds orprocedures that reduce pain in the animals can be readily tested byobserving behavioral characteristics of challenged animals in thepresence versus the absence of the test compounds) or procedure.

Exemplary behavioral tests used to study chronic pain include tests ofspontaneous pain, allodynia, and hyperalgesia. To assess spontaneouspain, posture, gait, nocifensive signs (e.g., paw licking, excessivegrooming, excessive exploratory behavior, guarding of the injured bodypart, and self-mutilation) can be observed. To measure evoked pain,behavioral-responses can be examined following exposure to heat (e.g.,thermal injury model).

Exemplary animal models of pain include, but are not limited to, theChung model, the carageenan induced hyperalgesia model, the Freund'scomplete adjuvant induced hyperalgesia model, the thermal injury model,the formalin model and the Bennett Model. The Chung model of neuropathicpain (without inflammation) involves ligating one or more spinal nerves.Chung et al. (2004) Methods Mol Med. 99: 35-45; Kim and Chung (1992)Pain 50: 355-363. Ligation of the spinal nerves results in a variety ofbehavioral changes in the animals including heat hyperalgesia, coldallodynia, and ongoing pain. Compounds that antagonize TRPA1 can beadministered to ligated animals to assess whether they diminish theseligation-induced behavioral changes in comparison to that observed inthe absence of compound.

Carageenan induced hyperalgesia and Freund's complete adjuvant (CFA)induced hyperalgesia are models of inflammatory pain. Walker et al.(2003) Journal of Pharmacol Exp Ther 304: 56-62; McGaraughty et al.(2003) Br J Pharmacol 140: 1381-1388; Honore et al. (2005) J PharmacolExp Ther. Compositions that inhibit TRPA1 can be administered tocarrageenan or CFA challenged animals to assess whether they diminishthermal hyperalgesia in comparison to that observed in the absence ofcompound. In addition, the ability of compounds that antagonize TRPA1function to diminish cold and/or mechanical hypersensitivity can also beassessed in these models. Typically, the carrageenan inducedhyperalgesia model is believed to mimic acute inflammatory pain and theCFA model is believed to mimic chronic pain and chronic inflammatorypain.

The Bennett model uses prolonged ischemia of the paw to mirror chronicpain. Xanthos et al. (2004) J Pain 5: S1. This provides an animal modelfor chronic pain including post-operative pain, complex regional painsyndrome, and reflex sympathetic dystrophy. Prolonged ischemia inducesbehavioral changes in the animals including hyperalgesia to mechanicalstimuli, sensitivity to cold, pain behaviors (e.g., paw shaking,licking, and/or favoring), and hyperpathia. Compositions that antagonizeTRPA1 can be administered to challenged animals to assess whether theydiminish any or all of these behaviors in comparison to that observed inthe absence of compound. Similar experiments can be conducted in athermal injury or UV-burn model which can be used to mimicpost-operative pain.

Additional models of neuropathic pain include central pain models basedon spinal cord injury. Chronic pain is generated by inducing a spinalcord injury, for example, by dropping a weight on a surgically exposedarea of spinal cord (e.g., weight-drop model). Spinal cord injury canadditionally be induced by crushing or compressing the spinal cord, bydelivering neurotoxin, using photochemicals, or by-hemisecting thespinal cord. Wang and Wang (2003).

Additional models of neuropathic pain include peripheral nerve injurymodels. The term peripheral neuropathy encompasses a variety ofdiseases, conditions, and injuries. One of skill in the art can readilyselect an appropriate model in light of the particular condition ordisease under investigation. Exemplary models include, but are notlimited to, the neuroma model, the Bennett model, the Seltzer model, theChung model (ligation at either L5 or L5/L6), the sciatic cryoneurolysismodel, the inferior caudal trunk resection model, and the sciaticinflammatory neuritis model. Exemplary models of inflammatory paininclude the rat model of intraplantar bradykinin injection. Briefly, thebaseline thermal sensitivity of the animals is assessed on a Hargreave'sapparatus. TRPA1 blockers are then administered systemically. Bradykininis subsequently injected into the paw and a hyperalgesia is. allowed todevelop. Thermal escape latency is then measured at multiple time pointsover the next few hours (Chuang et al., 2001; Vale et al., 2004).

Exemplary models of neuropathic pain associated with particular diseasesare also available. Diabetes and shingles are two diseases oftenaccompanied by neuropathic pain. Even following an acute shinglesepisodes, some patients continue to suffer from postherpetic neuralgiaand experience persistent pain lasting years. Neuropathic pain caused byshingles and/or postherpetic neuralgia can be studied in thepostherpetic neuralgia model (PHN). Diabetic neuropathy can be studiedin diabetic mouse models, as well as chemically induced models ofdiabetic neuropathy. Wang and Wang (2003).

Cancer pain has any of a number of causes, and numerous animal modelsexist to examine cancer pain related to, for example, chemotherapeuticsor tumor infiltration. Exemplary models of toxin-related cancer paininclude the vincristine-induced peripheral neuropathy model, thetaxol-induced peripheral neuropathy model, and the cisplatin-inducedperipheral neuropathy model. Wang and Wang (2003). An exemplary model ofcancer pain caused by tumor infiltration is the cancer invasion painmodel (CIP).

Primary and metastatic bone cancers are associated with tremendous pain.Several models of bone cancer pain exist including the mouse femur bonecancer pain model (FBC), the mouse calcaneus bone cancer pain model(CBC), and the rat tibia bone cancer model (TBC).

In addition to any of the foregoing models of chronic pain, compositionsthat inhibit TRPA1 function can be tested in one or more models of acutepain. Valenzano et al. (2005) Neuropharmacology 48: 658-672. Regardlessof whether compounds are tested in models of chronic pain, acute pain,or both, these studies are typically (though not exclusively) conducted,for example, in mice, rats, or guinea pigs. Additionally, compounds canbe tested in various cell lines that provide in vitro assays of pain.Wang and Wang (2003).

Many individuals seeking treatment for pain suffer from visceral pain.Animal models of visceral pain include the rat model of inflammatoryuterine pain (Wesselmann et al., (1997) Pain 73:309-317), injection ofmustard oil into the gastrointestinal tract to mimic irritable bowelsyndrome (Kimball et al., (2005) Am J Physiol Gastrointest LiverPhysiol, 288(6):G1266-73), injection of mustard oil into the bladder tomimic overactive bladder or bladder cystitis (Riazimand (2004), BJU. 94:158-163). The effectiveness of a TRPA1 compound can be assessed by adecrease in writhing, gastrointestinal inflammation or bladderexcitability.

The foregoing animal models are relied upon in the study of pain. Thefollowing provide additional exemplary references describing the use ofthese models in the study of pain: thermal injury model (Jones andSorkin, 1998, Brain Res 810: 93-99; Nozaki-Taguchi and Yaksh, 1998,Neuroscience Lett 254: 25-28; Jun and Yaksh, 1998, Anesth Analg 86:348-354), formalin model (Yaksh et al., 2001, J Appl Physiol 90:2386-2402), carrageenan-model (Hargreaves et al., 1988, Pain 32:-77-88),and CFA model (Nagakura et al., 2003, J Pharmacol Exp Ther 306:490-497).

Administration

It is preferable to administer the TRPA1 inhibitors of the invention asa pharmaceutical formulation (composition). The compositions accordingto the invention are formulated for administration in any convenient wayfor use in human or veterinary medicine. Regardless of the route ofadministration selected, the compositions of the present invention,which are used in a suitable hydrated form, and/or the pharmaceuticalagents of the present invention, are formulated into pharmaceuticallyacceptable dosage forms such as described below or by other conventionalmethods known to those of skill in the art.

Thus, another aspect of the present invention provides pharmaceuticalcompositions comprising a therapeutically effective amount of one ormore of the agents described herein, formulated together with one ormore pharmaceutically acceptable carriers (additives) and/or diluents.As described in detail below, in some examples, the pharmaceuticalcompositions of the present invention are specially formulated foradministration in solid or liquid foil, including those adapted for thefollowing: (1) oral administration, for example, drenches (aqueous ornon-aqueous solutions or suspensions), tablets, boluses, powders,granules, pastes for application to the tongue; (2) parenteraladministration, for example, by subcutaneous, intramuscular orintravenous injection as, for example, a sterile solution or suspension;(3) topical application, for example, as a cream, ointment or sprayapplied to the skin; (4) intravaginally or intrarectally, for example,as a pessary, cream or foam; or (5) for inhalation. However, in certainexamples the subject agents are simply dissolved or suspended in sterilewater. In certain examples, the pharmaceutical preparation isnon-pyrogenic, i.e., does not elevate the body temperature of a patient.

The phrase “therapeutically effective amount” as used herein means thatamount of a compound, material, or composition comprising a compound ofthe present invention which is effective for producing some desiredtherapeutic effect by inhibiting TRPA1 function in at least asub-population of cells in an animal and thereby blocking the biologicalconsequences of that function in the treated cells, at a reasonablebenefit/risk ratio applicable to any medical treatment.

The phrases “systemic administration,” “administered systemically,”“peripheral administration” and “administered peripherally” as usedherein mean the administration of a compound, drug or other materialother than directly into the central nervous system, such that it entersthe patient's system and, thus, is subject to metabolism and other likeprocesses, for example, subcutaneous administration.

The phrase “pharmaceutically acceptable” is employed herein to refer tothose compounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of human beings and animals without excessive toxicity,irritation, allergic response, or other problem or complication,commensurate with a reasonable benefit/risk ratio.

The phrase “pharmaceutically acceptable carrier” as used herein means apharmaceutically acceptable material, composition or vehicle, such as aliquid or solid filler, diluent, excipient, solvent or encapsulatingmaterial, involved in carrying or transporting the subject antagonistsfrom one organ, or portion of the body, to—another organ, or portion ofthe body. Each carrier must be “acceptable” in the sense of beingcompatible with the other ingredients of the formulation and notinjurious to the patient. Some examples of materials which can serve aspharmaceutically acceptable carriers include: (1) sugars, such aslactose, glucose and sucrose; (2) starches, such as corn starch andpotato starch; (3) cellulose, and its derivatives, such as sodiumcarboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4)powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients,such as cocoa butter and suppository waxes; (9) oils, such as peanutoil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil andsoybean oil; (10) glycols, such as propylene glycol; (11) polyols, suchas glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters,such as ethyl oleate and ethyl laurate; (13) agar; (14) bufferingagents, such as magnesium hydroxide and aluminum hydroxide; (15) alginicacid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer'ssolution; (19) ethyl alcohol; (20) phosphate buffer solutions; and (21)other non-toxic compatible substances employed in pharmaceuticalformulations.

Wetting agents, emulsifiers and lubricants, such as sodium laurylsulfate and magnesium stearate, as well as coloring agents, releaseagents, coating agents, sweetening, flavoring and perfuming agents,preservatives and antioxidants can also be present in the compositions.

Formulations of the present invention include those suitable for oral,nasal, topical (including buccal and sublingual), rectal, vaginal and/orparenteral administration. The formulations can be conveniently bepresented in unit dosage form and prepared by any methods well known inthe art of pharmacy. The amount of active ingredient which can becombined with a carrier material to produce a single dosage form willvary depending upon the host being treated, the particular mode ofadministration. The amount of active ingredient that can be combinedwith a carrier material to produce a single dosage form will generallybe that amount of the compound which produces a therapeutic effect.Generally, out of one hundred percent, this amount will range from about1 percent to about ninety-nine percent of active ingredient, preferablyfrom about 5 percent to about 70 percent, most preferably from about 10percent to about 30 percent.

Formulations of the invention suitable for oral administration are inthe form of capsules, cachets, pills, tablets, lozenges (using aflavored basis, usually sucrose and acacia or tragacanth), powders,granules, or as a solution or a suspension in an aqueous or non-aqueousliquid, or as an oil-in-water or water-in-oil liquid emulsion, or as anelixir or syrup, or as pastilles (using an inert base, such as gelatinand glycerin, or sucrose and acacia) and/or as mouth washes and thelike, each containing a predetermined amount of a compound of thepresent invention as an active ingredient. In further examples,compositions of the present invention are also administered as a bolus,electuary or paste.

In solid dosage forms of the invention for oral administration(capsules, tablets, pills, dragees, powders, granules and the like), theactive ingredient is mixed with one or more pharmaceutically acceptablecarriers, such as sodium citrate or dicalcium phosphate, and/or any ofthe following: (1) fillers or extenders, such as starches, lactose,sucrose, glucose, mannitol, and/or silicic acid; (2) binders, such as,for example, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose and/or acacia; (3) humectants, such as glycerol;(4) disintegrating agents, such as agar-agar, calcium carbonate, potato,or tapioca starch, alginic acid, certain silicates, and sodiumcarbonate; (5) solution retarding agents, such as paraffin; (6)absorption accelerators, such as quaternary ammonium compounds; (7)wetting agents, such as, for example, cetyl alcohol and glycerolmonostearate; (8) absorbents, such as kaolin and bentonite clay; (9)lubricants, such a talc, calcium stearate, magnesium stearate, solidpolyethylene glycols, sodium lauryl sulfate, and mixtures thereof; and(10) coloring agents. In the case of capsules, tablets and pills, thepharmaceutical compositions also comprise buffering agents in someexamples. Solid compositions of a similar type also can be employed asfillers in soft and hard-filled gelatin capsules using such excipientsas lactose or milk sugars, as well as high molecular weight polyethyleneglycols and the like.

A tablet comprising agents of the invention can be made by compressionor molding, optionally with one or more accessory ingredients.Compressed tablets are prepared using binder (for example, gelatin orhydroxypropylmethyl cellulose), lubricant, inert diluent, preservative,disintegrant (for example, sodium starch glycolate or cross-linkedsodium carboxymethyl cellulose), surface-active or dispersing agent.Molded tablets are made by molding in a suitable machine a mixture ofthe powdered compound moistened with an inert liquid diluent.

The tablets, and other solid dosage forms of the pharmaceuticalcompositions of the present invention, such as dragees, capsules, pillsand granules, are optionally be scored or prepared with coatings andshells, such as enteric coatings and other coatings well known in thepharmaceutical-formulating art. They are also formulated so as toprovide slow or controlled release of the active ingredient thereinusing, for example, hydroxypropylmethyl cellulose in varying proportionsto provide the desired release profile, other polymer matrices,liposomes and/or microspheres. They are sterilized by, for example,filtration through a bacteria-retaining filter, or by incorporatingsterilizing agents in the form of sterile solid compositions that can bedissolved in sterile water, or some other sterile injectable mediumimmediately before use. These compositions are also optionally containopacifying agents and are of a composition that they release the activeingredient(s) only, or preferentially, in a certain portion of thegastrointestinal tract, optionally, in a delayed manner. Examples ofembedding compositions that can be used include polymeric substances andwaxes. The active ingredient can also be in micro-encapsulated form, ifappropriate, with one or more of the above-described excipients.

Liquid dosage forms for oral administration of the compounds of theinvention include pharmaceutically acceptable emulsions, microemulsions,solutions, suspensions, syrups and elixirs. In addition to the activeingredient, the liquid dosage forms contain inert diluents commonly usedin the art, such as, for example, water or other solvents, solubilizingagents and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethylcarbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propyleneglycol, 1,3-butylene glycol, oils (in particular, cottonseed, groundnut,corn, germ, olive, castor and sesame oils), glycerol, tetrahydrofurylalcohol, polyethylene glycols and fatty acid esters of sorbitan, andmixtures thereof.

Besides inert diluents, the oral compositions can also include adjuvantssuch as wetting agents, emulsifying and suspending agents, sweetening,flavoring, coloring, perfuming and preservative agents.

Alternatively or additionally, compositions can be formulated fordelivery via a catheter, stent, wire, or other intraluminal device.Delivery via such devices are especially useful for delivery to thebladder, urethra, ureter, rectum, intestine, or intrathecal delivery,for example.

Formulations of the present invention which are suitable for vaginaladministration also include pessaries, tampons, creams, gels, pastes,foams or spray formulations containing such carriers as are known in theart to be appropriate.

Dosage forms for the topical or transdermal administration of a compoundof this invention include powders, sprays, ointments, pastes, creams,lotions, gels, solutions, patches and inhalants. The active compoundsare mixed under sterile conditions with a pharmaceutically acceptablecarrier, and with any preservatives, buffers, or propellants that arerequired.

The ointments, pastes, creams and gels contain, in addition to an activecompound of this invention, excipients, such as animal and vegetablefats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives,polyethylene glycols, ‘silicones, bentonites, silicic acid, talc andzinc oxide, or mixtures thereof.

Powders and sprays can contain, in addition to a compound of thisinvention, excipients such as lactose, talc, silicic acid, aluminumhydroxide, calcium silicates and polyamide powder, or mixtures of thesesubstances. Sprays can additionally contain customary propellants, suchas chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons,such as butane and propane.

Transdermal patches have the added advantage of providing controlleddelivery of a compound of the present invention to the body. Such dosageforms can be made by dissolving or dispersing the compound in the propermedium. Absorption enhancers can also be used to increase the flux ofthe compound across the skin. The rate of such flux can be controlled byeither providing a rate controlling membrane or dispersing the compoundin a polymer matrix or gel.

Ophthalmic formulations, eye ointments, powders, solutions and the like,are also contemplated as being within the scope of this invention.

The phrases “parenteral administration” and “administered parenterally”as used herein means modes of administration other than enteral andtopical administration, usually by injection, and includes, withoutlimitation, intravenous, intramuscular, intraarterial, intrathecal,intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal,transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular,subarachnoid, intraspinal and intrasternal injection and infusion.

Pharmaceutical compositions of this invention suitable for parenteraladministration comprise one or more compounds of the invention incombination with one or more pharmaceutically acceptable sterileisotonic aqueous or nonaqueous solutions, dispersions, suspensions oremulsions, or sterile powders which are reconstituted into sterileinjectable solutions or dispersions just prior. to use, which containantioxidants, buffers, bacteriostats, solutes which render theformulation isotonic with the blood of the intended recipient orsuspending or thickening agents.

Examples of suitable aqueous and nonaqueous carriers that are employedin the pharmaceutical compositions of the invention include water,ethanol, polyols (such as glycerol, propylene glycol, polyethyleneglycol, and the like), and suitable mixtures thereof, vegetable oils,such as olive oil, and injectable organic esters, such as ethyl oleate.Proper fluidity can be maintained, for example, by the use of coatingmaterials, such as lecithin, by the maintenance of the required particlesize in the case of dispersions, and by the use of surfactants.

These compositions also contain adjuvants such as preservatives, wettingagents, emulsifying agents or dispersing agents in some examples.Prevention of the action of microorganisms are ensured by the inclusionof various antibacterial and antifungal agents, for example, paraben,chlorobutanol, phenol sorbic acid, and the like. In some examples, it isdesirable to include isotonic agents, such as sugars, sodium chloride,and the like into the compositions. In addition, prolonged absorption ofthe injectable pharmaceutical form is brought about by the inclusion ofagents that delay absorption such as aluminum monostearate and gelatin.

In some cases, in order to prolong the effect of a drug, it is desirableto slow the absorption of the drug from subcutaneous or intramuscularinjection. This is accomplished by the use of a liquid suspension ofcrystalline or amorphous material having poor water solubility. The rateof absorption of the drug then depends upon its rate of dissolution,which, in turn, depend upon crystal size and crystalline form.Alternatively, delayed absorption of a parenterally administered drugform is accomplished by dissolving or suspending the drug in an oilvehicle.

Injectable depot forms are made by forming microencapsule matrices ofthe subject compounds in biodegradable polymers such aspolylactide-polyglycolide. Depending on the ratio of drug to polymer,and the nature of the particular polymer employed, the rate of drugrelease can be controlled. Examples of other biodegradable polymersinclude poly(orthoesters) and poly(anhydrides). Depot injectableformulations are also prepared by entrapping the drug in liposomes ormicroemulsions that are compatible with body tissue.

When the compositions of the present invention are administered aspharmaceuticals, to humans and animals, they can be given per se or as apharmaceutical composition containing, for example, 0.1 to 99.5% (morepreferably, 0.5 to 90%) of active ingredient in combination with apharmaceutically acceptable carrier.

The addition of the active compound of the invention to animal feed ispreferably accomplished by preparing an appropriate feed premixcontaining the active compound in an effective amount and incorporatingthe premix into the complete ration.

Alternatively, an intermediate concentrate or feed supplement containingthe active ingredient can be blended into the feed. The way in whichsuch feed premixes and complete rations can be prepared and administeredare described in reference books (such as “Applied Animal Nutrition”,W.H. Freedman and CO., San Francisco, U.S.A., 1969 or “Livestock Feedsand Feeding” O and B books, Corvallis, Ore., U.S.A., 1977).

Methods of introduction are also provided by rechargeable orbiodegradable devices. Various slow release polymeric devices have beendeveloped and tested in vivo in recent years for the controlled deliveryof drugs, including proteinacious biopharmaceuticals. A variety ofbiocompatible polymers (including hydrogels), including bothbiodegradable and non-degradable polymers, can be used to form animplant for the sustained release of a compound at a particular targetsite.

Actual dosage levels of the active ingredients in the pharmaceuticalcompositions of this invention are varied so as to obtain an amount ofthe active ingredient that is effective to achieve the desiredtherapeutic response for a particular patient, composition, and mode ofadministration, without being toxic to the patient.

The selected dosage level will depend upon a variety of factorsincluding the activity of the particular compound of the presentinvention employed, or the ester, salt or amide thereof, the route ofadministration, the time of administration, the rate of excretion of theparticular compound being employed, the duration of the treatment, otherdrugs, compounds and/or materials used in combination with theparticular compound employed, the age, sex, weight, condition, generalhealth and prior medical history of the patient being treated, and likefactors well known in the medical arts. A physician or veterinarianhaving ordinary skill in the art can readily determine and prescribe theeffective amount of the pharmaceutical composition required. Forexample, the physician or veterinarian could start doses of thecompounds of the invention employed in the pharmaceutical composition atlevels lower than that required in order to achieve the desiredtherapeutic effect and gradually increase the dosage until the desiredeffect is achieved. Ultimately, the attending physician or veterinarianwill decide the appropriate amount and dosage regimen.

In general, a suitable daily dose of a compound of the invention will bethat amount of the compound that is the lowest dose effective to producea therapeutic effect. Such an effective dose will generally depend uponthe factors described above. Generally, intravenous, intracerebroventricular and subcutaneous doses of the compounds of this inventionfor a patient will range from about 0.0001 to about 100 mg per kilogramof body weight per day.

If desired, the effective daily dose of the active composition areadministered as two, three, four, five, six or more sub-dosesadministered separately at appropriate intervals throughout the day,optionally, in unit dosage forms.

The patient receiving this treatment is any animal in need, includingprimates, in particular humans, and other mammals such as equines,cattle, swine and sheep; and poultry and pets in general.

The compound of the invention can be administered as such or inadmixtures with pharmaceutically acceptable and/or sterile carriers andcan also be administered in conjunction with other antimicrobial agentssuch as penicillins, cephalosporins, aminoglycosides and glycopeptides.Conjunctive therapy thus includes sequential, simultaneous and separateadministration of the active compound in a way that the therapeuticeffects of the first administered one are still detectable when thesubsequent therapy is administered.

The present invention contemplates formulation of the subject compoundsin any of the aforementioned pharmaceutical compositions andpreparations. Furthermore, the present invention contemplatesadministration via any of the foregoing routes of administration.

One of skill in the art can select the appropriate formulation and routeof administration based on the condition being treated and the overallhealth, age, and size of the patient being treated.

Combination Therapy

Another aspect of the invention provides a combination therapy whereinone or more other therapeutic agents are administered with the TRPA1inhibitors described herein. Such combination treatment is achieved byway of the simultaneous, sequential, or separate dosing of theindividual components of the treatment. In certain examples, acomposition of the invention is conjointly administered with ananalgesic. Suitable analgesics include, but are not limited to, opioids,glucocorticosteroids, non-steroidal antiinflammatories,naphthylalkanones, oxicams, para-aminophenol derivatives, propionicacids, propionic acid derivatives, salicylates, fenamates, fenamatederivatives, pyrozoles, and pyrozole derivatives. Examples of suchanalgesic compounds include, but are not limited to, codeine,hydrocodone, hydromorphone, levorpharnol, morphine, oxycodone,oxymorphone, butorphanol, dezocine, nalbuphine, pentazocine, etodolac,indomethacin, sulindac, tolmetin, nabumetone, piroxicam, acetaminophen,fenoprofen, flurbiprofen, ibuprofen, ketoprofen, naproxen, diclofenac,oxaprozin, aspirin, diflunisal, meclofenamic acid, -mefanamic acid,prednisolone, and dexamethasone. Preferred-analgesics are non-steroidalantiinflammatories and opioids (preferably morphine).

In certain examples, a composition of the invention is administered incombination with a non-steroidal anti-inflammatory. Suitablenon-steroidal antiinflammatory compounds include, but are not limitedto, piroxicam, diclofenac, etodolac, indomethacin, ketoralac, oxaprozin,tolmetin, naproxen, flubiprofen, fenoprofen, ketoprofen, ibuprofen,mefenamic acid, sulindac, apazone, phenylbutazone, aspirin, celecoxiband rofecoxib.

In certain examples, a composition of the invention is administered incombination with an antiviral agent. Suitable antiviral agents include,but are not limited to, amantadine, acyclovir, cidofovir, desciclovir,deoxyacyclovir, famciclovir, foscamet, ganciclovir, penciclovir,azidouridine, anasmycin, amantadine, bromovinyldeoxusidine,chlorovinyldeoxusidine, cytarbine, didanosine, deoxynojirimycin,dideoxycitidine, dideoxyinosine, dideoxynucleoside, edoxuidine,enviroxime, fiacitabine, foscamet, fialuridine, fluorothymidine,floxuridine, hypericin, interferon, interleukin, isethionate,nevirapine, pentamidine, ribavirin, rimantadine, stavirdine,sargramostin, suramin, trichosanthin, tribromothymidine,trichlorothymidine, vidarabine, zidoviridine, zalcitabine3-azido-3-deoxythymidine, 2′,3-dideoxyadenosine (ddA),2′-,3′-dideoxyguanosine (ddG), 2′,3′-dideoxycytidine (ddC),2′,3′-dideoxythymidine (ddT), 2′3′-dideoxy-dideoxythyrnidine (d4T),T-deoxy-3′-thia-cytosine (3TG or lamivudime),2t,3′-dideoxy-2′-fluoroadenosine, 2′,3′-dideoxy-2′-fluoroinosine,2′,3′-dideoxy-2′-fluorothymidine, 2′,3′-dideoxy-2′-fluorocytosine,2l3′-dideoxy-2I,3l-didehydro-2′-fluorothymidine (Fd4T),2′3′-dideoxy-2′-beta-fluoroadenosine (F-ddA),2′3′-dideoxy-2′-beta-fluoro-inosine (F-ddl), and2′53′-dideoxy-2′-beta-flurocytosine (F-ddC), trisodiumphosphomonoformate, trifluoro thymidine, 3′azido-3′thymidine (AZT),dideoxyinosine (ddl), and idoxuridine.

In certain examples, a composition of the invention is conjointlyadministered with an antibacterial agent. Suitable antibacterial agentsinclude, but are not limited to, amanfadine hydrochloride, amanfadinesulfate, amikacin, amikacin sulfate, amoglycosides, amoxicillin,ampicillin, amsamycins, bacitracin, beta-lactams, candicidin,capreomycin, carbenicillin, cephalexin, cephaloridine, “cephalothin,cefazolin, cephapirin, cephradine, cephaloglycin, chilomphenicols,chlorhexidine, chloshexidine gluconate, chlorhexidine hydrochloride,chloroxine, chlorquiraldol, chlortetracycline, chlortetracyclinehydrochloride, ciprofloxacin, circulin, clindamycin, clindamycinhydrochloride, clotrimazole, cloxacillin, demeclocycline,diiodohydroxyquin, doxycycline, ethambutol, ethambutol hydrochloride,erythromycin, erythromycin estolate, erhmycin stearate, farnesol,floxacillin, gentamicin, gentamicin sulfate, gramicidin, giseofulvin,haloprogin, haloquinol, hexachlorophene, iminocylcline,iodochlorhydroxyquin, kanamycin, kanamycin sulfate, lincomycin,lineomycin, lineomycin hydrochloride, macrolides, meclocycline,methacycline, methacycline hydrochloride, methenine, methenaminehippurate, methenamine mandelate, methicillin, metonidazole, miconazole,miconazole hydrochloride, minocycline, minocycline hydrochloride,mupirocin, nafcillin, neomycin, neomycin sulfate, netimicin, netilmicinsulfate, nitromrazone, norfloxacin, nystatin, octopirox, oleandomycin,orcephalosporins, oxacillin, oxyteacline, oxytetracycline hydrochloride,parachlorometa xylenol, paromomycin, paromomycin sulfate, penicillins,penicillin G, penicillin V, pentamidine, pentamidine hydrochloride,phenethicillin, polymyxins, quinolones, streptomycin sulfate,tetracycline, tobramycin, tolnaftate, triclosan, trifampin, rifamycin,rolitetracycline, spectinomycin, spiramycin, struptomycin, sulfonamide,tetracyclines, -tetracycline, tobramycin, tobramycin sulfate,triclocarbon, triclosan, trimethoprim-sulfamethoxazole, tylosin,vancomycin, and yrothricin. In certain examples, a compound of theinvention is conjointly administered with a cough suppressant,decongestant, or expectorant.

Examples of retinoids that be administered with the subject TRPA1inhibitors, e.g., where the TRPA1 inhibitor can be used to reduce thepain and/or inflammatory effect of the retinoid, include, but are notlimited to, compounds such as retinoic acid (both cis and trans),retinol, adapalene, vitamin A and tazarotene. Retinoids are useful intreating acne, psoriasis, rosacea, wrinkles and skin cancers and cancerprecursors such as melanoma and actinic keratosis.

Similarly, the subject TRPA1 inhibitors can be used in conjunction withkeratolytic agents include benzoyl peroxide, alpha hydroxyacids, fruitacids, glycolic acid, salicylic acid, azelaic acid, trichloroaceticacid, lactic acid and piroctone.

The subject TRPA1 inhibitors can be used with anti-acne agents,anti-eczema agents and anti-psoratic agents. The subject TRPA1inhibitors can also be used with skin protectants, such allantoin andesculin.

In certain examples, two or more compostions of the invention areadministered in combination. When two or more compositions of theinvention are conjointly administered, the two or more compositions havea similar selectivity profile and functional activity, or the two ormore compounds have a different selectivity profile and functionalactivity. By way of example, the two or more compositions are bothapproximately 10, 100, or 1000 fold selective for antagonizing afunction of TRPA1 over TRPV1, TRPV5, and TRPV6 (e.g., the two or morecompounds have a similar selectivity profile), and further inhibit afunction of TRPA1 with a similar TC50 (e.g., a similar functionalactivity). Alternatively, the one of the two or more compositionsselectively inhibit TRPA1 while the other of the two or morecompositions inhibits both TRPA1 and TRPV1 (e.g., the two or morecompounds have differing selectivity profiles). Administration ofcombinations of two or more compounds of the invention having similar ordiffering properties are contemplated. In certain examples, acomposition of the invention is conjointly administered with one or moreadditional compounds that antagonize the function of a differentchannel. By way of example, a composition of the invention is conjointlyadministered with one or more compounds that antagonize TRPV1, TRPM8,and/or TRPV3. The compound(s) that antagonize TRPV1, TPRM8, or TRPV3 areselective for TRPV1, TRPM8 or TRPV3 (e.g., inhibit TRPV1 or TRP V3 10,100, or 1000 fold more strongly than TRPA1). Alternatively, thecompound(s) that antagonize TRPV1 or TRP V3 cross react with other TRPchannels.

In certain other examples, a composition of the invention is conjointlyadministered with one or more additional agents or therapeutic regimensappropriate for the particular injury, disease, condition, or disorderbeing treated.

Kits

The present invention also features kits comprising a Tmem100 mutantpolypeptide, for example a Tmem100 mutant polypeptide that comprises SEQID NO: 1, or fragments thereof or SEQ ID NO: 3, or fragments thereof.

The kit also contains instructions for providing the Tmem100 mutantpolypeptide, or fragment thereof, to a cell, for example a sensoryneuron.

The kit contains instructions for use in preventing, treating, oralleviating symptoms of pain or preventing, treating, or alleviatingsymptoms of itch.

EXAMPLES

The discovery of Pirt provides a model for the regulation of TransientReceptor Potential (TRP) channel multi-subunit complexes. It isexpressed in more than 90% of dorsal root ganglia (DRG) neurons and hasbeen identified as a positive regulator of TRPV1 (Kim et al., 2008) andTRPM8 (Tang et al., 2013). Initially, Pirt was identified by a cDNAsubtractive screen using neonatal wild-type and Ngn1−/− mouse dorsalroot ganglion (DRG) to find genes specifically expressed in nociceptiveneurons (Dong et al., 2001). The Pirt protein independently formscomplexes with TRPV1 and TRPM8, augmenting the responses of thesechannels to their agonists. At the behavioral level, pain associatedwith these TRP channels is also reduced. Pirt −/− mice demonstratedattenuated responses to noxious stimuli such as heat, cold, andchemicals like capsaicin and icilin. Pirt −/− mice also revealed adrastic deficiency in the itch responses evoked by a wide array ofpruritogens (Patel et al., 2011). Nevertheless, there is a lack ofevidence showing that Pirt is able to regulate the activity of TRPA1(Kim et al., 2008). Here studies describe other Pirt-like proteins andpeptides that regulate TRPs and modify their functions in a variety ofTRP channel complexes.

Using the protein sequence of Pirt, another gene has been identified,Tmem100, with the working name of Tmem100 given its similar structuraland biochemical properties. Like Pirt, Tmem100 is a 134 amino-acidtwo-transmembrane protein with both N- and C-termini being intracellularand an amino acid sequence that is highly conserved in vertebrates (Moonet al., 2010). Thus, both Pirt and Pirt2 (i.e., Tmem 100) have similarprotein size, membrane topology, and function, i.e., regulating TRPchannels in DGR neurons. Unlike the restricted expression pattern ofPirt, Tmem100 is expressed more widely in other organs besides the DRG.It is expressed as early as embryonic day 9.5 (E9.5) in the dorsal aortaand from E10.5 to E12.5 in other vessels, ventral neural tubes, and thenotochord (Moon et al., 2010). It is reported to be associated withrenal development (Georgas et al., 2009), vasculogenesis (Moon et al.,2010; Somekawa et al., 2012), lung cancer cell invasiveness (Frullantiet al., 2012), body height in African-Americans (Carty et al., 2012),and apoptosis (Yamazaki et al., 2011). However, little is known aboutthe underlying mechanisms of these Tmem100-associated effects and itsrole in the nervous system.

A large body of evidence indicates that TRP channels are capable ofassembling into heterotetrameric channel complexes. This phenomenon wasoriginally reported for TRPC channels: TRPC1 co-assembles with TRPC4 andTRPC5 in the rat brain (Strübing et al., 2001). In mammals, theformation of various TRP channel complexes containing channels from theTRPC (Goel et al., 2002; Hofmann et al., 2002; Strubing et al., 2003),TRPV (Hellwig et al., 2005; Rutter et al., 2005; Smith et al., 2002),TRPM (Chubanov et al., 2004), and TRPP (Schaefer, 2005) families havebeen demonstrated. Thus, TRPs are able to heteromerize within the samesubfamily (e.g., TRPV1 and TRPV3) (Cheng et al., 2012) and acrosssubfamilies (e.g., TRPV4 and TRPP2) (Kottgen et al., 2008). The subunitcomposition influences the biophysical and regulatory properties of theresulting channel complex (Xu et al., 1997). They have uniquebiophysical and pharmacological properties and are modulated viadistinct pathways (Cheng et al., 2012). It was demonstrated that theTRPA1-TRPV1 (TRPA1-V1) complex is present in sensory neurons with uniquebiophysical properties (Salas et al., 2009; Staruschenko et al., 2010).Since TRPA1 and TRPV1 contribute significantly to peripheral and centralmechanisms of pain and hypersensitivity (Julius, 2013), the TRPA1-V1complex could be clinically important. In addition, regulation of thisTRP channel complex could provide specificity in the management of painand hypersensitivity in various pathophysiological conditions.

The present work identifies Tmem100 as a potentiating modulator ofTRPA1-V1 complexes. Tmem100 is co-expressed with TRPA1 and TRPV1 inpeptidergic DRG neurons. Tmem100-deficient mice show a reduction ininflammatory hypersensitivity and TRPA1-but not TRPV1-mediated pain.Single-channel recording in a heterologous system reveals that Tmem100selectively potentiates TRPA1 activity in the TRPA1-V1 complex in aTRPV1-dependent manner. Mechanistically, Tmem100 weakens the associationof TRPA1 and TRPV1 and thereby releases the inhibition of TRPA1 byTRPV1. A Tmem100 mutant, Tmem100-3Q, exerts the opposite effect, i.e. itenhances the association of TRPA1 and TRPV1 and strongly inhibits TRPA1.Strikingly, a cell permeable peptide (CPP) sharing the C-terminalsequence of Tmem100-3Q mimics its effect in the presence of TRPV1,selectively inhibiting TRPA1-mediated pain. The studies described hereinunveil a context-dependent modulation of the TRPA1-V1 complex, andTmem100-3Q CPP represents a novel therapeutic for pain management.

Studies propose a putative TRPA1-V1 complex model in which a membraneadapter protein—Tmem100—regulates the physical association between TRPA1and TRPV1 (FIG. 8). Further studies demonstrate that TRPA1 activity isinhibited by TRPV1 when the two channels are co-expressed in the absenceof Tmem100 (FIGS. 4A, 8A) (Salas et al., 2009). When Tmem100 is present,TRPA1 activity is potentiated in a TRPV1-dependent fashion (FIGS. 2A-2J;4A-4E). The presence of Tmem100 weakens the TRPA1-V1 association byphysical interaction with both channels (FIGS. 5E, 8B), which results indisinhibition of TRPA1 and a net positive effect on TRPA1-associatedactivity in the TRPA1-V1 complex. Moreover, Tmem100-3Q (with strongerinteraction with TRPV1 and no interaction with TRPA1; FIGS. 5 and13A-13D) exerts the opposite effect, tightening the physical associationbetween TRPA1 and TRPV1 (FIG. 5E); TRPA1 inhibition by TRPV1 isincreased (FIGS. 8C, 8D). The uniqueness of Tmem100 is that it not onlyprovides a feasible model of regulation for TRPA1-V1 complexes but alsodemonstrates selectivity and context dependency.

Tmem100 preferentially augments the responses to TRPA1 agonists in theTRPA1-V1 complex whereas the responses to TRPV1 agonists remainrelatively unchanged. This phenomenon is consistent from single channelsall the way to the behavioral level. Electrophysiological andbiochemical results indicate that Tmem100 is able to physicallyassociate with TRPA1 and TRPV1 alone and modulate them. However, theseregulatory effects are inhibitory in nature for both TRPA1 and TRPV1homomers. Interestingly, the inhibitory effects of Tmem100 were notobserved in DRG neurons and behavioral assays. One possible explanationis that most TRPA1⁺ DRG neurons also express TRPV1; therefore, theamount of TRPA1 homomer could be minimal in DRO neurons in either normalor pathological conditions (Diogenes et al., 2007). An alternatepossibility is that Tmem100 modulates channels other than the TRPA1-V1complex and contributes to other phenotypes (Somekawa et al., 2012).Data from a heterologous expression system suggested that Tmem100inhibit CAP-mediated responses in TRPV1⁺/TRPA1⁻ DRG neurons. Oneexplanation is that Tmem100 has low expression levels in theTRPV1⁺/TRPA1⁻ neuronal subset (FIG. 1). Studies will test thispossibility.

The discovery of Tmem100 also reconciles the inconsistent effects ofTRPV1 on TRPA1-mediated currents reported in native sensory neuronsversus heterologous systems (Salas et al., 2009). MO-induced currentswere smaller in sensory neurons from TRPV1^(−/−) mice. This is incontrast to the expected increase in currents since TRPV1 inhibits TRPA1in heterologous systems. Data described herein provides an explanationfor the underlying mechanisms. In Tmem100^(−/−) DRG neurons, as seen inthe heterologous system, TRPA1-mediated activity was lowered compared toTmem100^(+/+) neurons, presumably due to the inhibitory effect of TRPV1.Heterologous studies show that Tmem100 itself also inhibits TRPA1activity in the absence of TRPV1 (compare the “A1” and “A1+T100” columnsin FIGS. 4A and S4A). Since TRPA1⁺ sensory neurons from TRPV1^(−/−) micestill express Tmem100, the MO-gated currents would be inhibited byTmem100. This inhibition is exactly what previous studies (Salas et al.,2009) and data provided herein (compare the open bars in FIGS. 7D and7H) have found in TRPV1^(−/−) DRG neurons and mice. Therefore, takinginto account the modulatory action of Tmem100, the data collected fromheterologous cells and native neurons are now consistent and suggestthat the interaction between TRPV1 and TRPA1 leads to an inhibition ofTRPA1 by TRPV1.

Several lines of evidence indicate that Tmem100 plays an important rolein pain and inflammation. First, treatment with NSAIDs andimmunosuppressants reduces its expression (Yamazaki et al., 2011).Second, inflammatory pain is reduced in Tmem100 sensory neuron-specificknockout mice. Third, Tmem100 is exclusively expressed in peptidergicDRG neurons, which are crucial for neurogenic inflammation (FIG. 1H).Lastly, TRPA1 and TRPV1, both key targets of Tmem100 modulation, arecritical regulators of inflammatory pain (Bautista et al., 2006;Caterina et al., 2000). Thus, an approach that interferes with thispathway will provide specificity to control pain under pathologicalconditions and will consequently be important in pain management.

The discovery and subsequent characterization of the T100-Mutcell-permeable peptide is an avenue for the management of pain. Theextent of the inhibitory effect of T100-Mut is comparable to that ofother potent TRPA1 antagonists (da Costa et al., 2010; McNamara et al.,2007; Petrus et al., 2007). The major advantage of this approach is tomaximize the specificity and thus minimize the possible side effects ofdrugs by specifically targeting the TRPA1-V1 complex instead of TRPhomomers expressed in other tissues. Therefore, studies described andproposed herein on Tmem100 lead to a better understanding of theprocessing, modulation, and management of pain.

Example 1: Materials and Methods Generation of Tmem100 GFP Knock-inMouse Line

Full-length Tmem100 cDNA from mouse DRG was cloned into the pGEM T-Easyvector (Promega) and later subcloned into the expression vectorpcDNA3.1. The arms of the Tmem100 targeting constructs were subclonedfrom ES cell genomic DNA. The gene deletion constructs eliminated exon3, which contains the entire coding region of Tmem100. PmeI and AscIrestriction sites were engineered so the EGFPf-ACN cassette could beplaced in the middle of the two arms. A negative selection marker (DTA)was placed outside of the right arm to increase homologous recombinationrate. Tmem100^(GFP/+) mice were generated using the targeting constructby the Transgenic Core Laboratory at the Johns Hopkins University.Generation of the mutant allele and excision of the ACN cassette wereverified by PCR and Southern blotting.

Generation of Tmem100 Conditional Knockout Mice

A BAC clone containing the entire Tmem100 genomic sequence was purchasedfrom Children's Hospital Oakland Research Institute and modified byrecombineering (Liu et al., 2003). The final gene targeting vectorcontains exon 3 and a PGK-Neo cassette flanked by two loxP sites. Thetwo homologous arms are 1.9 and 6.0 kb in size, respectively.Tmem100^(fl/+) mice were generated using the targeting construct by theTransgenic Core Laboratory at the Johns Hopkins University. Homologousrecombination in ES cells was verified by PCR for both arms with a longrange PCR kit (Roche, 04829034001) and sequencing. Germline transmissionwas confirmed by PCR. The F1 progeny were mated with FlpE mice toeliminate the PGK-Neo cassette, and the results were verified with PCRtargeting the Neo cassette. For the behavioral studies, mice werecrossed with WT C57/BL6 for more than 5 generations before mating to anAvil^(+/CRE) line.

Western Blot of DRG Lysate

DRG from cervical to lumbar levels were collected in 300 μL PBS and 2%SDS with protease inhibitor (Sigma P8340). After sonication, thesolution was centrifuged at 18,000 rcf for 5 minutes in 4 degrees. Thesupernatants were collected and stored at −80 degrees. For the westernblots, DRG lysates were separated by SDS-PAGE (10% or 15% for Tmem100detection) and wet transferred to PVDF membranes (GE Healthcare,RPN303F). The membranes were blocked with 5% milk in TBST for 30minutes. The primary antibodies used were: 1:5000 anti-Tmem100, 1:1000anti-TRPV1 (Santa Cruz, R130), and 1:1000 anti-TRPA1 (Novus,NB110-40763). Secondary antibodies for visualization included donkeyanti-rabbit and anti-mouse HRP-conjugated antibodies (GE Biosciences).The intensities for the signals were analyzed using ImageJ.

Co-Immunoprecipitation (Co-IP)

DRG from all levels or CHO cells transfected with Trpv1, Trpa1, andTmem100-myc were used. Whole cell DRG or CHO cells lysates weregenerated 24 h after transfection, and Co-IP with either 1 μg mycantibody (EMD Millipore) or 1 μg TRPV1 antibody (Santa Cruz, R130) asdescribed (Akopian et al., 2007) Immunoprecipitants and cell lysatealiquots were resolved by SDS-PAGE and immunoblotted with 1:1000anti-TRPV1 antibody (Santa Cruz, R130), 1:500 anti-Tmem100 antibody,1:1000 anti-TRPA1 (Novus), or 1:1000 anti-myc (EMD Millipore). Secondaryantibodies for visualization included donkey anti-rabbit and anti-mouseHRP-conjugated antibodies (GE Biosciences).

GST Pull-Down

The GST-N, GST-C fusion, and GST constructs (2 μg) were individuallytransfected with 2 μg of Trpa1, Trpv1, Trpm8, and Trpv2 constructs intoHEK293T cells with Lipofectamine 2000. Twenty-four hours later, cellswere washed with PBS and lysed with 500 μL. IP buffer (1% TritonX-100+protease inhibitor in PBS). After sonication and centrifugationfor 10 min at 13,000 rpm at 4° C., the supernatants were incubated withglutathione-agarose beads (GE bioscience). The bound proteins wereeluted from the beads by heating in 2× protein sample buffer at 50° C.for 10 minutes. The samples were resolved by SDS-PAGE and immunoblottedwith 1:2500 rabbit polyclonal anti-TRPV1 antibody (Santa Cruz, R130),1:1000 rabbit anti-TRPA1 antibody (Novus), 1:5000 anti-GST antibody(Sigma A7360), or 1:10000 donkey anti-rabbit HRP-conjugated antibody (GEBioscience NA934V).

Behavioral Assays Hot Plate Methods

Mice were placed in a clear plexiglass cylinder on top of atemperature-controlled metal plate (Life Science Series 8, Model 39.)The latency of acute nocifensive responses was determined by the onsetof hindpaw lifts and/or licking, flinching, or jumping.

Tail Immersion Test

Mice were restrained in an apparatus made of 50 mL conical tubes. Theirtails were exposed in the water bath set to the designated temperatures.

Von Frey Methods

Mice were placed in a transparent plastic box (4.5×5×10 cm) on a metalmesh and acclimatized for 30 minutes prior to testing. Each mouse wastested more than 5 times at a specific force manually, and the thresholdwas determined by the lowest force needed to elicit responses more than50% of the time.

Mustard Oil Injection

Mice were injected intradermally in the hind paw with 6 μL of 0.2%mustard oil (Sigma-Aldrich 377430) with Hamilton needles (80300). Themice were placed in a plexiglass cylinder, and the total time spentlicking and flinching was recorded for the first 10 minutes afterinjection. Thirty and sixty minutes after injection, the mice wereassayed with von Frey filaments for mechanical hyperalgesia.

Hargreaves Test

Mice were placed under a transparent plastic box (4.5×5×10 cm) on aglass platform (Plantar Test Apparatus, IITC Life Science). Radiant heatwas adjusted to 18% of maximal output and shone on the center of thepaws. Each mouse was tested more than 3 times, with each test performed20 minutes apart.

CFA Injection

Mice were injected with 6 μL of 50% emulsified Complete Freund'sAdjuvant (Sigma, F5881) in normal saline.

Capsaicin Injection

Mice were injected with 6 μL of capsaicin (0.1 μg/μL in normalsaline/10% ethanol/0.5% Tween 80) in the hindpaws. The mice were placedin a plexiglass cylinder, and the total time spent licking and flinchingwas recorded for the first 10 minutes after injection.

Paclitaxel Injection

Mice were injected with paclitaxel (Sigma T7191) intraperitoneally atthe dose of 6 mg/kg, as previously described (Materazzi et al., 2012).Seven days after paclitaxel injection, the mice were assayed with VonFrey methods for mechanical hyperalgesia, both before and four hoursafter injection of cell permeable peptides.

Cold Plate Test

A metallic plate on a bed of ice was cooled in a −20° C. freezer. Duringthe test, the plate was allowed to warm to 0° C. as measured by thetemperature probe. The onset of brisk hindpaw lifts and/orflicking/licking of the hindpaw was assessed.

Rat L5 SNL

An modified L5 SNL model was produced as described in previously (He etal., 2014; Shechter et al., 2013). Male Sprague-Dawley rats (200-350 g,Harlan, Indianapolis, Ind.) were anesthetized with 2% isoflurane. Theleft L5 spinal nerve was ligated with a 6-0 silk suture and cutdistally. The muscle layer was closed with 4-0 chromic gut suture andthe skin closed with metal clips. For intrathecal catheter implantation,a small slit was cut in the atlanto-occipital membrane of rats, intowhich a saline-filled piece of PE-10 tubing (6-7 cm) was inserted (He etal., 2014). After completing the experiment, it was confirmedintrathecal drug delivery by injecting lidocaine (400 μg/20 μl, Hospira,Lake Forest, Ill.), which resulted in a temporary motor paralysis of thelower limbs. Hypersensitivity to punctuate mechanical stimulation wasdetermined with the up-down method by using a series of von Freyfilaments (0.38-15.1 g) applied for 4-6 seconds to the test area on theplantar surface of the hindpaw (Chaplan et al., 1994; He et al., 2014).The PWT was determined according to the formula provided by Dixon(Dixon, 1980). Rats that underwent SNL but did not develop mechanicalhypersensitivity (>50% reduction of PWT from pre-SNL baseline) by day 5post-SNL and rats that showed impaired motor function or deterioratinghealth after treatment were eliminated from the subsequent behavioralstudies, and data were not analyzed. HC-030031 was purchased from TocrisBioscience (Bristol, UK). Both drugs were dissolved in 10%dimethylsulfoxide (DMSO), 5% Tween 80 and 85% sterile saline solution.The final working solution which was injected by intrathecal routecontained <1% DMSO. The number of animals used in each study was basedon experience with similar studies and power analysis calculations.Animals were randomized to the different treatment groups and blindedthe experimenter to drug treatment to reduce selection and observationbias. After the experiments were completed, no data point was excluded.STATISTICA 6.0 software (StatSoft, Inc., Tulsa, Okla.) was used toconduct all statistical analyses. The Tukey honestly significantdifference (HSD) post-hoc test was used to compare specific data points.Bonferroni correction was applied for multiple comparisons. Two-tailedtests were performed, and data are expressed as mean±SEM; P<0.05 wasconsidered significant in all tests.

Calcium Imaging

Calcium imaging assays were performed as previously described (Liu etal., 2009). Cells were loaded with 2 μM fura 2-acetomethoxy ester(Molecular Probes) for 30 min in the dark at room temperature or for 45minutes at 37° C. for DRG and cell lines, respectively. After washing,cells were imaged at 340 and 380 nm excitation to detect intracellularfree calcium under a fluorescent microscope (Nikon Eclipse TE2000-S) andLambda 10B shutter (Sutter Instrument). The cells were bathed in calciumimaging buffer (pH 7.45, 130 mM NaCl, 3 mM KCl, 2.5 mM CaCl₂, 10 mMHEPES, 10 mM glucose, 1.2 mM NaHCO₃, with sucrose to increase osmolarityto 290 mOsm). The reagents and buffers were applied through a gravityperfusion system at a rate of 2 mL/s, including mustard oil (Sigma377430), cinnamaldehyde (Sigma C80687), menthol (Sigma M2780), andcapsaicin (Sigma M2028). Cells with high baseline 340/380 values (>1.5)were excluded for analysis. For DRG neurons, IB₄ staining was performedin the chamber with the dilution of 1:200 (Molecular Probes 121412) for160 seconds before washing off at the end of each test. Images wereprocessed and analyzed with NIS-Elements BR 2.30 software (Nikon). Aresponsive cell was defined as one that with greater than a 20% increasein the 340/380 ratio above the baseline. Intracellular calibration forcalcium was performed as previously described (Akopian et al., 2007).The data was analyzed with the experimenter blinded to the genotypes orconstructs transfected.

Cell Culture

DRG from 3 to 4-week old mice were collected in cold DH10 medium (90%DMEM/F-12, 10% FBS, 100 U/ml penicillin, and 100 μg/ml Streptomycin,Gibco) and treated with enzyme solution in HBSS containing collagenase(1.65 mg/mL, Worthington CLS I) and dispase (3.55 mg/mL, GIBCO17105-041) at 37° C. for 30 minutes. After trituration andcentrifugation, cells were resuspended in DH10, plated on glasscoverslips coated with poly-D-lysine (0.5 mg/ml, Stoughton, Mass.) andlaminin (10 μg/ml, Invitrogen), and cultured overnight in an incubatorat 37° C. The neurons were tested within 24 hours. HEK293T, COS-7, andCHO cells were cultured in a medium consisting of 90% DMEM, 10% FBS, 100U/mL penicillin, and 100 μg/ml Streptomycin. GlutaMAX (Gibco 35050-061)was also added for CHO cells. For calcium imaging, the cells were platedon glass coverslips coated with poly-D-lysine (0.5 mg/ml, Stoughton,Mass.). In the HEK cell studies the denominator is the cells expressingboth TRPA1, TRPV1, and the designated constructs (i.e. Tmem100 orTmem100-3Q mutant). mCherry:TRPV1:Tmem100 was transfected in 1:8:8 ratiointo TRPA1 stable cell line (obtained from N. Tigue fromGlaxoSmithKline) and used mCherry as the marker for the cells expressingthe constructs 18 hours after transfection with Lipofectamine 2000. Thetransfection efficiency for Tmem100+TRPV1+TRPA1 triple-positive cells is27±3%. This efficiency was determined by mCherry(+) divided by totalnumber of cells in the bright fields.

Electrophysiology

Recordings were made in cell-attached single-channel or whole-cellvoltage clamp configurations at 22-24° C. from the somata ofsmall-to-medium mouse DRG neurons (15-35 pF) or CHO cells. Forwhole-cell configuration, holding potential (Vh) is −60 mV. Data wereacquired and analyzed using an Axopatch 200B amplifier and pCLAMP9.0software (Molecular Devices, Sunnyvale, Calif.). Borosilicate pipettes(Sutter, Novato, Calif.) were pulled and polished to resistances of 3-5MΩ (in whole-cell and single-channel pipette solutions). Accessresistance (Rs) was compensated (40-80%) when appropriate up to thevalue 7-10 MΩ for whole-cell configuration. Whole-cell recording datawere filtered at 0.5 kHz and sampled at 2 kHz. Single-channels currentswere filtered with an 8-pole, low pass Bessel filter at 0.1 kHz andsampled at 0.5 kHz, since dwelling time (τ) of the TRPA1 and TRPV1single-currents were >0.5 sec. Whole-cell recorded data were rejectedwhen Rs changed >20% during recording, leak currents were >100 pA, orinput resistance was <200 MΩ Currents were considered positive whentheir amplitudes were 5-fold bigger than displayed noise (in root meansquare).

Single-channel unitary current (i) was determined from the best-fitGaussian distribution of amplitude histograms. Single-channel activitywas analyzed as NPo=I/i, where I is the mean total current in a patchand i is unitary current at this voltage. Open probability (Po) for mainconductance is presented in figures. For single channel slopeconductances, linear fitting was used separately for positive andnegative holding potentials. The slope conductances presented in figureswere determined from fitting negative hold potentials (from −60 to 0mV). The single-channel recording data were analyzed with Clampfit 9software. This has an “event detection analysis” function that analyzescurrent traces and determines the number of channels in the patch (N),how many sub-conductances are present, and what the main conductance is.In order to increase the accuracy of the Po measurement, only patchescontaining fewer than 3 channels were used. For patches containing bothTRPA1 and TRPV1, only those with equal numbers of TRPA1 and TRPV1 wereused. This was monitored in each recording by applying MO and then CAP.

Standard external solution (SES) for whole-cell patch recordingcontained (in mM): 140 NaCl, 5 KCl, 2 CaCl₂. 1 MgCl₂, 10 D-glucose and10 HEPES, pH 7.4. Vehicle, which is 0.01% DMSO, was added to externalsolutions. The bath solution for single-channel recording (SES-SCh) ofTRP currents consisted of (in mM): 100 K-gluconate, 4 KCl, 1 MgCl₂, 1EGTA, 10 D-glucose and 10 Hepes (pH 7.3). The standard pipette solution(SIS) for the whole-cell configurations contained (in mM): 140 KCl, 1MgCl₂, 1 CaCl₂, 10 EGTA, 10 D-glucose, 10 HEPES, pH 7.3. The pipettesolution for single-channel recording (SIS-SCh) was (mM): 100Na-gluconate, 10 NaCl, 1 MgCl₂, 2 CaCl₂, 10 D-glucose and 10 HEPES (pH7.3). Drugs were applied using a fast, pressure-driven, computercontrolled 8-channel system (ValveLink8; AutoMate Scientific, SanFrancisco, Calif.). The baseline activities of the cells were recordedfor 1-2 min prior drug applications. The durations of drug applicationsare noted in legends to figures. Single-channel analyses of traces wereperformed from the 10th to 30th seconds after commencing drugapplications.

Immunofluorescent Staining

Adult mice of 8-12 weeks old were anesthetized with pentobarbital andperfused with 20 ml 0.1 M phosphate-buffered saline (PBS; pH 7.4; 4degrees) followed with 25 ml 4% paraformaldehyde and 14% picric acid inPBS (4 degrees). After perfusion, spinal cords and dorsal root ganglia(DRG) were dissected. DRG was post-fixed in 4% paraformaldehyde and 14%picric acid at 4 degrees for 30 min and spinal cord was fixed for 1 hr.All tissues were cryoprotected in 20% sucrose in PBS at 4 degreesovernight, and preserved in OCT at −80 degree. Before staining, twentyμm sections were post-fixed with 4% paraformaldehyde in PBS for 10minutes and washed with 0.1% PBST. After blocking in 10% goat serum inPBST for 1 hour at room temperature, sections were incubated withprimary antibodies at 4° C. overnight. The primary antibodies used were:rabbit anti-GFP (Invitrogen, 1:1000), chicken anti-GFP (Invitrogen,1:1000), rabbit anti-CGRP (Bachem T-4239, 1:1000), rabbit anti-NF200(Sigma N4142, 1:1000), rabbit anti-TRPV1 (gift from Dr. Caterina,1:1000), IB₄ (Molecular Probes 1-21412, 1:200), mouse anti-NeuN(Millipore MAB377, 1:300), and rabbit anti-TRPA1 (gift from Dr. Schmidt,1:200). On the second day, the sections were incubated with secondaryantibodies at room temperature for 1 hour. The secondary antibodies usedwere: goat anti-rabbit (Invitrogen A11036, Alexa 568-conjugated; A11034,Alexa 488-conjugated), goat anti-chicken (Invitrogen A11039, Alexa488-conjugated), goat anti-rat (Invitrogen A11434, Alexa555-conjugated), and goat anti-mouse IgG1 (Invitrogen A-21124, Alexa568-conjugated; A-21121, Alexa 488 conjugated). All secondary antibodieswere diluted 1:300 in the blocking solution. Sections were washed withPBST and mounted with Fluoromount-G (Southern BioTech). Images wereobtained using the Zeiss LSM700 confocal microscope system.

Live Staining

F11 cell line was transfected with 0.6 μg of eitherpcDNA_(3.1)-Tmem100-myc or pcDNA_(3.1)-Tmem100 +0.2 μg mCherry followingLipofectamine 2000 protocol. On the second day, the cells weretrypsinized and plated on 150 mm coverslips coated with poly-D-lysine.The non-detergent-treated (NDT) groups were washed 3 times with bufferconsisting of 1% goat serum in PBS. The detergent-treated (DT) groupswere fixed with 4% PFA in PBS for 15 minutes at room temperature andwashed with buffer consisting of 1% goat serum in 0.1% PBST. Afterwashing, the coverslips were blocked in 10% goat serum in theirrespective washing buffers for 15 minutes at room temperature. They wereincubated with primary antibodies (1:200 mouse anti-c-myc ab, 9B11, 1mg/mL) or 1:1000 rabbit anti-Tmem100 antibody for 1 hour at roomtemperature and washed 3 times. They were incubated with secondaryantibodies (1:1000 goat anti-mouse IgG-488 (Invitrogen) or goatanti-rabbit IgG-488 (Invitrogen)) for 30 min at room temperature.

Total Internal Reflection Fluorescence (TIRF) Microscopy and ForsterResonance Energy Transfer (FRET)

Expression vectors of pEYFP-TRPA1 (YFP on C-terminal part), pECFP-TRPV1(CFP on C-terminal part), and pEYFP-N1 were transfected into COS-7 cellswith FuGENE HD (Promega E2311), as previously described (Staruschenko etal., 2010). COS-7 cells were chosen since they have flat morphology andthus suitable for TIRF-FRET analysis. Moreover, previously it has beenshown that CHO and COS cells express TRPA1 and TRPV1 to the same level(Staruschenko et al., 2010). Data from fixed cells were collected inseparate facilities at University of Texas Health Science Center, SanAntonio, and the Johns Hopkins University, respectively. Each group wasco-transfected with full-length pcDNA_(3.1)-Tmem100,pcDNA_(3A)-Tmem100-3Q, or pcDNA_(3.1)-myc/His and plated on glass-bottomdishes (MatTek, P35G-1.0-14-C). FRET was essentially performed aspreviously described (Staruschenko et al., 2010). Briefly, two daysafter transfection, the cells were fixed for 15 min with 4%paraformaldehyde in PBS and then imaged at the room temperature usingtotal internal reflection fluorescence (TIRE) (also calledevanescent-field) microscopy on an inverted Nikon Eclipse TE200Umicroscope equipped with a plain Apo TIRF 60× oil-immersion,high-resolution (1.45 NA) objective. The CFP and YFP fluorophores wereexcited with a 442-nm Melles Griot dual-pulsed solid state and 514-nmargon ion laser, respectively, with an acoustic optic tunable filterused to select excitation wavelengths (Prairie Technology, Middleton,Wis.). Emissions from CFP and YFP passed through an image splittingdevice (Dual-View, Optical Insights, Tucson, Ariz.) using a 505-nmdichroic filter to split emissions, which then passed through 470±15 and550±25 nm emission filters, respectively. Fluorescence images werecollected and processed with a 16-bit, cooled charge-coupled devicecamera (Cascade 512F; Roper Scientific Inc.) interfaced to a PC runningMetamorph software.

Each cell was photobleached by argon-ion laser (514 nm) at full powerfor 2 min. It was demonstrated previously preferential photo-bleachingof membrane proteins in and near the plasma membrane abutting thecoverglass versus total cellular pools of the channel with TIRFillumination (Staruschenko et al., 2010). The % FRET efficiency wascalculated as the percent increase in CFP after photobleaching:

% FRET=(CFPpost−CFPpre)/CFPpost

where CFPpost and CFPpre are the mean grey values of CFP emission in thecells after and before photobleaching subtracted by its background,respectively. All images were analyzed in ImageJ.

Cell Permeable Peptides

The sequence from the last 28 amino acids of the C-terminus of theTmem100-3Q mutant protein was synthesized and myristoylated at itsN-terminus (myr-WKVRQRNKKVQQQESQTALVVNQRCLFA-COOH) (SEQ ID NO: 8) byTwenty first Century Biochemicals. The scrambled peptide was synthesizedwith the same composition and did not resemble any known protein(myr-QRVLEQVLQNWSRRANVKQAQKFQVKCT-COOH) (SEQ ID NO: 15). For the calciumimaging assay, the peptides were added to the calcium imaging bufferwith a final concentration of 200 nM and incubated for at least 30 minat room temperature. For the mice behavioral assays, the peptides (5 μL,2 mM) were injected subcutaneously in the hindpaw at least 30 minutesbefore testing. For the rat behavior assays, human T100-3Q (h-T100), apalmitoylated cell permeable peptide mutated based on the C terminus ofhuman sequence in Tmem100, was applied. The sequence is Palmitoyl-

(SEQ ID NO: 7) WKVRQRSKKAQQQESQTALVANQRSLFA-COOH.

Statistical Analysis

Error bars are presented as mean±SEM. Numerical data in the text ispresented as mean±SEM. n represents the number of mice, individualresponding cells or individual tests analyzed. Statistical comparisonsbetween two groups were conducted by two-tailed, unpaired Student's ttest. Multiple groups were compared and analyzed by using one-way ANOVAand Bonferroni's post-hoc test (where each column was compared to allother columns). Differences between groups with genotype and time asfactors were accessed by two-way ANOVA with Bonferroni's multiplecomparison post-hoc tests. Power analysis was used to justify the samplesize. Differences were considered as statistically significant forp<0.05. Representative data are from experiments that were replicatedbiologically at least three times with similar results.

Example 2: Tmem100 Encodes a Two-Transmembrane Protein Expressed inPeptidergic DRG Neurons

Topology of Tmem100 was invested to understand its cellular localizationand distribution. Protein structure analysis (PredictProtein, ColumbiaUniversity, and SOSUI, Nagoya University, Japan) indicates that Tmem100is a two-transmembrane protein (FIG. 1A). Tmem100-myc construct wastransfected with c-myc at the C-terminus into the F11 cell line andstained with anti-myc antibody. Tmem100 was visualized at the plasmamembrane only after membrane permeabilization (FIGS. 9A,C). Similarresults were obtained with anti-Tmem100 antibody against the N-terminus(FIG. 9B). Staining also showed that the signal was primarily located inthe plasma membrane (FIG. 9D). The data indicate that Tmem100 is atwo-transmembrane protein largely localized to the plasma membrane withintracellular localization of both N- and C-termini.

To characterize Tmem100-expressing DRG neurons, a knock-in line wasgenerated in which the open reading frame of Tmem100 was replaced withGFP (Tmem100^(GFP/+); FIGS. 9E,F). Anti-Tmem100 antibody labellingconfirmed that GFP is specifically expressed in Tmem100⁺ DRU neurons inthe Tmem100^(GFP/+) mouse line (FIG. 9F). Using GFP as a marker, it wasfound that Tmem100 was expressed in 24% of lumbar DRG neurons (mainlysmall and medium in size) (FIG. 1B). Different DRG neuron markers weredouble stained with GFP in the Tmem100^(GFP/+) line (FIGS. 1C,D, and9G). Ninety-five percent of Tmem100⁺ neurons express CGRP, and 88.4% ofCGRP neurons express Tmem100. Both TRPA1 and TRPV1 are co-expressed in asubset of Tmem100⁺ neurons. In contrast, Tmem100⁺ neurons were rarelypositive for IB₄ (FIGS. 1C,D, and 11). These results suggest thatTmem100 is primarily expressed in peptidergic DRG neurons (FIG. 1E),many of which are TRPV1 and TRPA1 double-positive (FIG. 1F) (Bautista etal., 2006; Story et al., 2003). A recent study has shown that TRPA1 isfunctionally expressed in IB4⁺ non-peptidergic neurons (Barabas et al.,2012). The culture conditions of dissociated DRG neurons can influencethe expression of TRPA1. On the other hand, the sensitivity ofanti-TRPA1 antibody misses the expression of TRPA1 in IB4⁺ neurons.Moreover, it was found that a significant increase in the number ofTmem100-expressing neurons in the DRG under inflammatory conditionsinduced by complete Freund's Adjuvant (CFA) injection (FIG. 9H,I).Collectively, the expression data suggest that Tmem100 is involved inthe modulation of pain.

Example 3: Selective Elimination of Tmem100 in Sensory Neurons Leads toa Reduction of Mechanical Hyperalgesia and TRPA1-Associated Nociception

Conditional knockout mice were generated to study the function ofTmem100 in the nociceptive pathway (Tmem100^(fl/fl); FIG. 2A) because aglobal knockout of Tmem100 is lethal at E10.5 (Moon et al., 2010).Tmem100^(fl/fl) mice were mated with male Advillin^(+/CRE) (Avil-Cre)mice to selectively eliminate Tmem100 in primary sensory neurons of theDRG (Hasegawa et al., 2007). These mice were viable, and there were noobvious differences in gross appearance and behavior among wild-type(WT), Avil-Cre;Tmem100^(+/+), Tmem100^(fl/fl), andAvil-Cre;Tmem100^(fl/fl) mice. The deletion of Tmem100 protein in theDRG was verified by immunofluorescent staining and Western blotting witha rabbit anti-Tmem100 antibody (FIGS. 2B and 10A). No significantchanges in TRPA1 and TRPV1 expression were observed in the DRO whenTmem100 was eliminated (FIGS. 10A-C). Moreover, developmental andmorphological phenotypes were evaluated in the DRG and spinal cord fromAvil-Cre; Tmem100^(GFP/fl) lines, and the results did not suggest anyrelated defects (FIGS. 10D-F).

Next it was determined whether Tmem100 plays a role in nociception andhyperalgesia/allodynia by performing behavioral tests on Tmem100 DRGconditional knockout mice, i.e., Avil-Cre;Tmem100^(fl/fl) (Tmem100 CKO).These mice exhibited normal mechanical sensitivity under naiveconditions (FIG. 2C). However, Tmem100 CKO mice exhibited reduced acutenocifensive behaviors induced by mustard oil (MO; an agonist of TRPA1(Bautista et al., 2006; Kwan et al., 2006)) compared toAvil-Cre;Tmem100^(+/+) and Tmem100^(fl/fl) controls (FIGS. 2D and 10G).TRPA1-dependent mechanical hyperalgesia generated by injection of MOinto mouse hindpaws (Bautista et al., 2006) was also significantlydecreased in Tmem100 CKO mice (FIG. 2E). The average threshold formechanically induced pain was reduced to 0.06 g in both control groupsand was significantly higher (0.34 g) in Tmem100 CKO mice. In theinflammatory pain model generated by injection of CFA into the hindpaws,Tmem100 CKO mice also showed attenuated mechanical hyperalgesia (FIG.2G), consistent with a previous report on the involvement of TRPA1 ininflammatory mechanical hyperalgesia (Petrus et al., 2007). Themechanical nociceptive threshold was reduced to 0.18 g and 0.11 g inAvil-Cre;Tmem100^(+/+) and Tmem100^(fl/fl) mice, respectively, but was0.43 g in Tmem100 CKO mice at day 2. These data show that deletion ofTmem100 in the DRG leads to a substantial reduction of inflammatorymechanical hyperalgesia and acute TRPA1-associated nociception.

Interestingly, TRPV1-associated acute nociceptive behavior andhyperalgesia remained relatively unperturbed in Tmem100 CKO mice.Tmem100 CKO mice did not show any significant deficits incapsaicin-induced acute nocifensive behavior in the hindpaw (FIG. 2F).Tail immersion and hot plate tests also failed to reveal any deficits inthe mutant mice (FIGS. 2I and 2J). Furthermore, CFA-induced thermalhyperalgesia, which is almost completely reversed after TRPV1 deletionbut unaltered after pharmacological blockade of TRPA1 (Caterina et al.,2000; Petrus et al., 2007), was also unchanged in Tmem100 CKO mice (FIG.2H). Cold-induced pain was also tested in these animals, and the resultssuggest that this modality is not affected by the elimination of Tmem100(FIG. 10H,I).

Example 4: TRPA1-Mediated Responses are Selectively Attenuated in DRGNeurons from Tmem100 CKO Mice

To investigate the function of Tmem100 at a cellular level, cultured DRGneurons from Tmem100 CKO mice were examined with calcium imaging andwhole-cell electrophysiology recording. The results showed a selectivereduction in TRPA1-but not TRPV1- or TRPM8-mediated activities in theDRG neurons when Tmem100 was deleted. In calcium imaging, onlyIB4-negative DRG neurons were analyzed since the majority of IB4⁺neurons do not express Tmem100 (in cultured conditions, only 4.8±0.8%IB4⁺ neurons expressed Tmem100, and 5.2±0.5% of Tmem100⁺ neurons wereIB4⁺; >900 neurons from 3 mice were analyzed for each marker). Thepercentage of DRG neurons responsive to MO and the alternativeTRPA1-specific agonist cinnamaldehyde (CA) was significantly lower whenTmem100 was eliminated (Bandell et al., 2004) (FIGS. 3A and 11A).Fourteen and 17% of DRO neurons from Avil-Cre;Tmem100^(+/+) mice (i.e.controls) showed responses to MO and CA, respectively, and thepercentages dropped to 6% and 8% in the Tmem100 CKO group. Conversely,the percentage of DRG neurons with TRPV1 activity remained unchanged as31% of IB4-negative DRG neurons responded to capsaicin in bothAvil-Cre;Tmem100^(+/+) and Tmem100 CKO mice. Menthol was also used as acontrol and observed no difference in the percentage of neuronsresponsive to menthol, which is mostly mediated by TRPM8 at thisconcentration (Bautista et al., 2006; Dhaka et al., 2007), between thecontrol and Tmem100 CKO neurons (FIG. 3A).

Whole-cell patch clamp recording of DRG neurons was carried out toinvestigate regulation of TRPA1 and TRPV1-mediated currents by Tmem100.The currents evoked by 7 and 25 μM MO (based on the dose-response curvein FIG. 11B) in capsaicin (CAP)-responsive neurons were significantlysmaller in DRG neurons from Tmem100 CKO mice compared toAvil-Cre;Tmem100^(+/+) neurons (FIGS. 3B,C). This finding also showedthat Tmem100 enhances TRPA1 activity.

The effect of Tmem100 on TRPV1-mediated responses was also examined CAP(100 nM)-evoked currents in DRG neurons were not significantly differentbetween Avil-Cre;Tmem100^(+/+) and Tmem100 CKO mice (FIGS. 3D,E).Therefore, Tmem100 shows no modulatory effect on TRPV1 activity.

Example 5: Tmem100 Enhances TRPA1 Activity in Heterologous ExpressionSystems

Since both behavioral and cellular analyses suggest native Tmem100positively regulates TRPA1, next it was asked if it was possible toreproduce a similar effect in a heterologous system. To mimic thesituations in wild-type and Tmem100^(−/−) DRG neurons, co-expression ofTRPA1 and TRPV1 in CHO cells in the presence or absence of Tmem100 wasperformed. MO-evoked whole-cell current density and intracellularcalcium accumulation were significantly higher when Tmem100 was presentwith TRPA1 and TRPV1 (FIGS. 3F,G). A biotinylation assay revealed atrend of decreasing TRPA1 levels (not statistically significant) on theplasma membrane when wild-type Tmem100 was co-expressed with both TRPV1and TRPA1, whereas TRPV1 levels on the plasma membrane remainedunchanged (FIGS. 11D-F). Because the effect of Tmem100 on TRPA1trafficking to the plasma membrane is opposite that of its enhancementof channel activity, the true effect of Tmem100 on the whole cell areeven stronger if the lowering effect on membrane trafficking of theTRPA1 channel is taken into account.

To demonstrate that Tmem100's actions can be reproduced in analternative heterologous expression system, co-expression TRPA1 andTRPV1 in HEK293T cells was performed. Tmem100 rendered 30% and 20% ofcells responsive to CA and MO, respectively, whereas only 13% and 8% ofcells responded to the same agonist in the control group withoutTmem100. Interestingly, this enhancement was abolished when TRPV1 wasreplaced by TRPM8 (FIGS. 11G,H), suggesting TRPM8 does not have aninhibitory effect on TRPA1 as TRPV1 does. Moreover. Tmem100 also loweredthe EC₅₀ to CA by almost threefold compared to the control group (FIG.11J). These results suggest that Tmem100 selectively enhances TRPA1activity at the whole cell level. Importantly, this positive effectrequires the presence of TRPV1.

Example 6: Tmem100 Selectively Increases the Single-Channel OpenProbability of the TRPA1-V1 Complex to Mustard Oil but not Capsaicin

To analyze the effect of Tmem100 on TRPA1 and TRPV1 channel properties,cell-attached single-channel recordings were performed. Using CHO cellsexpressing various combinations of Tmem100. TRPA1, and TRPV1, openprobabilities (Po), single-channel activity (NPo), and conductance inresponse to MO and CAP were investigated. The presence of TRPA1-V1 inthe recording patch was confirmed with single-channel responses to bothCAP and MO. When Tmem100 was co-expressed with TRPA1 and TRPV1, asubstantial increase was seen in the TRPA1 Po; there was no increase inTRPA1 unitary single-channel conductance (FIGS. 4A,E 11K, and 12E). Thepresence of Tmem100 also caused a higher percentage of cells to respondto MO and CA (FIG. 11L,M). This modulation is not a dose-dependenteffect based on the ratio of Tmem100 to TRPA1 or TRPV1 since theexperiments with a different transfection ratio of Tmem100 still yieldedsimilar results (FIG. 11I). Interestingly, co-expression of Tmem100 withTRPA1 in the absence of TRPV1 significantly reduced the TRPA1 Po forboth the main conductance and unitary single-channel conductance (FIGS.4A,E, 12C, and 12E). Recordings of TRPV1 and TRPA1, in both heterologousexpression systems and sensory neurons, exhibit multiple single-channelsub-conductance states (Nagata et al., 2005; Premkumar et al., 2002;Staruschenko et al., 2010). This sub-conductance will influence thewhole-cell response; therefore, to examine the sub-conductancecontributions, the effects of Tmem100 on single-channel activity (NPo)of TRPA1 and TRPV1 were also analyzed. FIG. 12A illustrates that Tmem100regulates TRPA1 NPo in the same way it affects Po, i.e., Tmem100increases the TRPA1 NPo in the presence of TRPV1 and reduces itsactivity when TRPV1 is absent.

TRPV1 single-channel Po and unitary conductance, as assessed by theapplication of CAP, was relatively unaffected by Tmem100 when TRPA1 wasalso present in patches (FIGS. 4B,E, and 12F). However, Tmem100 reducedsingle-channel CAP responses in CHO cells expressing TRPV1 alone; thiseffect was much weaker in DRG neurons (FIG. 12K). Taken together, theseresults suggest that Tmem100 requires TRPV1 to increase intrinsicactivity of TRPA1. Tmem100 inhibits the intrinsic activity of individualTRPA1 and TRPV1 channels when the two are not co-expressed.

Example 7: Tmem100 Binds Both TRPA1 and TRPV1

To test the possibility that the functional interaction of Tmem100 withTRPA1 and TRPV1 is due to a complex assembled in sensory neurons,co-immunoprecipitation (co-IP) experiments from DRG lysates wereperformed. The results show that Tmem100 forms complexes with endogenousTRPA1 and TRPV1 in mouse DRG (FIG. 5A). Furthermore, to investigate thecontribution of each protein to complex formation, co-IP experimentsusing heterologous cells co-expressing Tmem100 with either TRPA1 orTRPV1 were carried out. Co-IP with full-length Tmem100-myc suggestedthat Tmem100 forms complexes with TRPV1 and TRPA1 individually (FIGS.5B,C). Glutathione S-tranferase (GST) pull-down studies using differentfragments of Tmem100 further characterized the distinct bindingproperties of its N- and C-termini. Both TRPA1 and TRPV1 separatelycould be pulled down with the C-terminal fragment of Tmem100; however,the same conditions with the N-terminus of Tmem100 only pulled downTRPV1 (FIG. 5D). These results indicate that Tmem100 can physicallyinteract with TRPA1 and TRPV1.

The K-R-R sequence in the C-terminus of Tmem100 was targeted because ofits positive charges and conservation in vertebrates (Moon et al.,2010). To investigate the functional significance of this sequence, itwas mutated to an uncharged Q-Q-Q sequence (Tmem100-3Q mutant).Interestingly, the Q-Q-Q mutation appeared to exert different effects onTRPA1 and TRPV1 binding. For TRPA1, the binding was abolished when theQ-Q-Q mutation was introduced to the C-terminus (GST-C-3Q). For TRPV1,this mutation did not appear to weaken binding but instead enhanced it(FIGS. 5D and 13A,B). These data show that although the C-terminus ofTmem100 binds to both TRPA1 and TRPV1, its binding sites to these twochannels are different. Therefore, Tmem100-3Q can exert distinctmodulatory effects on TRPA1 and TRPV1 compared to wild-type Tmem100.

Example 8: Wild-Type Tmem100 Weakens the Physical Association ofTRPA1-V1 Whereas Tmem100-3Q Enhances it

Förster resonance energy transfer (FRET) and total internal reflectionfluorescence (TIRF) microscopy were used to search for physical evidenceof the modulatory mechanisms of Tmem100 on the TRPA1-V1 complex. Cellsco-expressing TRPV1-CFP and TRPA1-YFP exhibited a significantly higherFRET efficiency than cells co-expressing TRPV1-CFP and membrane-tetheredYFP (“A1-V1” versus “M-V1” in FIG. 5E). This implies that TRPA1 andTRPV1 form a complex in the plasma membrane. Strikingly, wild-typeTmem100 weakened the surface TRPA1-V1 interaction, decreasing the FRETefficiency by 34% compared to the efficiency of cells expressing onlyTRPA1-V1. By contrast, the Tmem100-3Q mutant enhanced the TRPA1-TRPV1interaction by 43% (FIG. 5E,F). These data suggest that Tmem100modulates the physical interaction of the TRPA1/TRPV1 complex, withwild-type Tmem100 and Tmem100-3Q exerting opposite effects on theseinteractions. In a separate FRET-TIRF experiment, the physicalinteraction between TRPA1 and Tmem100 were found to be further augmentedin the presence of TRPV1 whereas that of TRPV1 and Tmem100 is unaffectedby TRPA1 (FIG. 13C,D). In addition, the interaction of TRPA1 and Tmem100is weaker than that of TRPV1 and Tmem100 (comparing “V1-T100” and“A1-T100” columns in FIG. 13C,D). The FRET data suggest preferentialinteractions between the three components. This result also shows thatthe physical interaction among the three proteins is not allostericinteraction, but occurs via specific interaction domains.

Example 9: Tmem100-3Q Selectively Inhibits TRPA1-Mediated Single ChannelActivity in the TRPA1-V1 Complex

Further functional investigation of Tmem100-3Q revealed that mutantTmem100 has the opposite effect of wild-type Tmem100 on TRPA1-mediatedsingle-channel properties at −60 mV (FIGS. 6A-E and 14A-D). It was foundthat in TRPA1-V1 co-expressing cells, Tmem100-3Q significantly decreasedsingle-channel MO-evoked Po with modest alterations in unitaryconductance (FIGS. 6A,E, and 14). Similarly, whole-cell MO-activatedcurrent density was significantly lower in TRPA1-V1 co-expressing cellscontaining Tmem100-3Q than control cells without Tmem100-3Q (FIG. 6F).Similar results were obtained by calcium imaging assay (FIG. 6G).Tmem100-3Q also lowered the percentage of cells that responded to MO andCA in HEK293T cells expressing both TRPA1 and TRPV1 (FIGS. 11L,M).Unlike MO responses in TRPA1-V1 cells, TRPV1-mediated single-channelactivity induced by CAP in TRPA1-V1 cells was relatively unchanged inthe presence of Tmem100-3Q (FIGS. 6B,D,E).

Like wild-type Tmem100, Tmem100-3Q also exerts context-dependentmodulatory effects on TRPA1 and TRPV1 homomers. Tmem100-3Q reducedsingle-channel responses of cells expressing TRPV1 alone whereas it hadno effect on TRPA1 responses in cells containing only TRPA1 (FIG. 6A,B).CAP responses from TRPV1-expressing CHO cells showed that the additionof Tmem100-3Q lowered its Po as well as NPo (FIGS. 6B, 14B,D). Incontrast, no modulatory effect of Tmem100-3Q on TRPA1-mediatedsingle-channel open probability (Po) or activity (NPo) was observed inTRPA1-expressing cells (FIGS. 6A, 14A,C). In summary, these data suggestthat Tmem100-3Q affects intrinsic and whole-cell TRPA1 activity only inthe presence of TRPV1.

Example 10: Cell-Permeable Peptides Mimicking Tmem100-3Q Attenuate TRPA1Activity and Block Pain

To selectively attenuate TRPA1-mediated activity in both cellular andbehavioral studies, a cell-permeable peptide was utilized, an approachthat has been employed to effectively modulate intracellular proteinactivity (Koren and Torchilin, 2012). A T100-Mut, a peptide that sharessequence with the C-terminus of Tmem100-3Q (FIG. 7A) was designed andsynthesized. In addition, the N-terminus of this peptide was conjugatedwith a myristoylated group, which allows peptides to penetrate andlocalize to the intracellular side of the plasma membrane (Nelson etal., 2007), thereby mimicking the topology of Tmem100-3Q (FIG. 8D).

At the cellular level, T100-Mut mimicked the effect of full-lengthTmem100-3Q by lowering TRPA1-mediated activity in the TRPA1-V1 complex.T100-Mut pretreatment selectively decreased responses to 10 μM MO inIB4-negative DRU neurons (FIG. 7B). However, the peptide had no effecton responses to 100 nM CAP in the same population (FIG. 7B). The resultswere largely consistent in a heterologous system. T100-Mut treatmentproduced a 65% reduction in the percentage of cells responding to MO(FIG. 7C). These results suggest that T100-Mut mainly reduced TRPA1-butnot TRPV1-associated activity. A CPP that shares sequence with thewild-type C terminus (T100-WT) was also tested. The result shows thatT100-WT does not have a modulatory effect on TRPA1-V1 complexes (FIG.7C).

Next, it was tested whether T100-Mut has a corresponding inhibitoryeffect on pain behaviors. T100-Mut-injected wild-type animals showedreduced MO-induced pain behavior (FIG. 7D). T100-Mut treatmentsignificantly decreased acute nocifensive behavior (36±6 s) compared tothe scrambled peptide-treated group (80±9 s). Mechanical hyperalgesiawas also significantly attenuated by T100-Mut (FIG. 7D). However,T100-Mut did not alter acute nocifensive behavior induced by CAP (FIG.7F). Similar results were obtained in the CFA model: 3 hours afterT100-Mut treatment, mice showed attenuated mechanical but not thermalhyperalgesia (FIG. 7E). The mechanical thresholds were significantlyhigher in the T100-Mut-treated group. The level of thermal hyperalgesiaremained similar between these two groups. T100-Mut also alleviatedmechanical hyperalgesia induced by paclitaxel (Taxol), a commonly usedchemotherapeutic agent that produces TRPA1-dependent neuropathy as aside effect (Materazzi et al., 2012) (FIG. 7G). Furthermore, a shortenedCPP (called F2) consisting of the first 18 amino acids of the T100-Mutcould produce the same inhibitory effects whereas two other shortenedCPPs that spanned different regions of the T100-Mut C-terminus had noeffect (FIG. 15A,B). Together, these data suggest that the first 4 aminoacids of the Tmem100 C-terminus, i.e. “WKVR”, and the 3Q mutations areessential for the inhibitory effect of TMEM100-3Q on TRPA1 in theTRPA1/V1 complex and that this effect is likely caused by an enhancedinteraction of TMEM100-3Q with TRPV1. Furthermore, there was no effectof T100-Mut on MO-induced acute pain and mechanical hyperalgesia inTrpv1^(−/−) mice (FIGS. 7H,I). The effects of T100-Mut and HC-030031, adirect TRPA1 antagonist (Eid et al., 2008), were also compared and itwas found that T100-Mut showed comparable or better efficacy and potencyof inhibiting neuropathic pain as the TRPA1 antagonist (FIG. 15C,D).These behavioral data demonstrate that T100-Mut selectively alleviatesTRPA1-associated pain and that this effect is TRPV1-dependent.

Example 11: Tmem100 Mutant Peptide Blocks Ongoing Pain

Ongoing, or spontaneous, pain, which is most bothersome to patients, isdifficult to treat and difficult to study in animals. The conditionedplace preference test has been successfully developed to reveal thepresence of non-evoked, ongoing pain and pain relief based on thepreference of an animal for the context paired with a pain-relieftreatment. The conditioned place preference can also capture theaversive aspects of pain. Strikingly, many studies have shown that therodent conditioned place preference test accurately reflects theeffectiveness of human clinical treatments. Therefore, this test is apowerful approach to investigate the potential effectiveness of newtreatments and mechanisms for relief of ongoing pain. Two previousstudies have examined the role of TRPA1 in ongoing pain by theconditioned place preference test. However, in both studies, inhibitionof TRPA1 by antagonists strongly blocked evoked mechanical hyperalgesiabut did not block ongoing pain induced by chronic pain models. Theseresults highlight differential mechanisms of evoked and ongoing pain.Because the antagonists used in those studies (HC030031 and Chem5861528)target TRPA1 monomers, it is possible that T100-Mut can inhibit ongoingpain by modulating the TRPA1/V1 complex. This possibility was tested inrodents with SNL-induced ongoing pain using the conditioned placepreference test. Data showed that one 10-μl dose of 50 μM human T100-Mutpeptide (also called P2-Mut) intrathecally injected into SNL ratsinduced robust the conditioned place preference, suggesting thatT100-Mut indeed blocked ongoing pain; the scrambled peptide had noeffect (FIG. 16A). Systemic administration of gabapentin, an analgesicthat is known to alleviate neuropathic pain, was used as a positivecontrol (FIG. 16B).

Example 12. Tmem100 and Itch in Conditional Knockout Mouse Model

The current evidence suggest that Tmem100 plays a role in itch. Thereare itch phenotypes in Tmem100 conditional knockout mice: they havedeficits for the scratching responses by histamine injection on the back(FIG. 16). A wide array of pruritogens will be tested in calcium imagingon Tmem100-deficient DRG neurons and by behavioral assays on Tmem100conditional knockout mice to interrogate the extent of itch phenotype.

Example 13. Human Tmem100-30 CPP Alleviates Neuropathic Pain by NerveInjury

In the wild-type mice with spinal nerve ligation (SNL), mechanicalhyperalgesia is observed 6 days post ligation. Palmitoylated (Pal) CPPtailored to the C-terminus of human Tmem100-3Q alleviates neuropathicpain/mechanical allodynia in SNL. n=6, *p<0.05 at Day 6 4 hrs post CPPtreatment. Scrambled: open bar; human P2-Mut (T100-Mut): black bar (FIG.17). Peptides (5 μl of 2 mM) were injected intradermally in thehindpaws. Sequences for CPP used in the SNL experiment:

Human P2-Mut (T100-Mut) Pal-WKVRQRSKKAQQQESQTALVANQRSLFA-OH (SEQ ID NO:7); Scrambled Pal-QRALEQVLQNWSRRANVKQAQKFQVKST-OH (SEQ ID NO: 19).Pal-palmitoylated in the N termini for each CPP.

Example 14: Functional Interaction Between TRPV1 and TRPA1

Functional interaction between TRPV1 and TRPA1 can occur in several ways(Julius, 2013) and TRPV1 and TRPA1 can modulate each other's activity byactivating intracellular pathways. For example, activation of TRPA1 cancause Ca²⁺ influx, triggering Ca²⁺-dependent phosphatases, which inturn, desensitize TRPV1 activity (Akopian et al., 2007). Alternatively,TRPA1-initiated Ca²⁺ influx could promote protein kinase A activity,sensitizing TRPV1 channels (Spahn et al., 2014). Behavioral experimentsdemonstrated that TRPA1 activation by MO leads to functionaldesensitization of TRPV1 responses in vivo (Jacquot et al., 2005;Ruparel et al., 2008). Similarly, activation of TRPV1 can lead toreduction of TRPA1 activity in vitro and in vivo (Akopian et al., 2007;Jacquot et al., 2005; Ruparel et al., 2008). An important element ofthese functional TRPV1-TRPA1 interactions is that the channels need tobe activated sequentially.

As described herein, an alternative mechanism could be the interactionof TRPV1 and TRPA1 within a complex. Indeed, evidence indicates that TRPchannels are capable of assembling into channel complexes (Schaefer,2005; Strubing et al., 2003). Physical interaction within TRP complexesalter the conformation of channels and thus influence the biophysicaland regulatory properties of each TRP channel (Xu et al., 1997). It wasdemonstrated that TRPV1 and TRPA1 can form a complex in heterologousexpression systems and sensory neurons (Akopian et al., 2007; Fischer etal., 2014; Staruschenko et al., 2010). Formation of such a complex canstrongly influence the properties of TRPA1 (Patil et al., 2011; Salas etal., 2009). Thus, as described herein, the composition and function ofTRPV1-TRPA1 complexes in sensory neurons is elucidated, as well as therequirement of TRPV1 activity for regulation within the complex.

INCORPORATION BY REFERENCE

All patents, published patent applications and other referencesdisclosed herein are hereby expressly incorporated by reference in theirentireties by reference.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents of the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

The recitation of a listing of elements in any definition of a variableherein includes definitions of that variable as any single element orcombination (or subcombination) of listed elements. The recitation of anembodiment herein includes that embodiment as any single embodiment orin combination with any other embodiments or portions thereof.

OTHER EMBODIMENTS

While the invention has been described in conjunction with the detaileddescription thereof, the foregoing description is intended to illustrateand not limit the scope of the invention, which is defined by the scopeof the appended claims. Other aspects, advantages, and modifications arewithin the scope of the following claims.

The patent and scientific literature referred to herein establishes theknowledge that is available to those with skill in the art. All UnitedStates patents and published or unpublished United States patentapplications cited herein are incorporated by reference. All publishedforeign patents and patent applications cited herein are herebyincorporated by reference. Genbank and NCBI submissions indicated byaccession number cited herein are hereby incorporated by reference. Allother published references, documents, manuscripts and scientificliterature cited herein are hereby incorporated by reference.

While this invention has been particularly shown and described withreferences to preferred embodiments thereof, it will be understood bythose skilled in the art that various changes in form and details may bemade therein without departing from the scope of the inventionencompassed by the appended claims.

1. A method for treating or preventing a condition associated with TRPA1function or for which reduced TRPA1 activity can reduce the severity,comprising administering an effective amount of a Tmem100 mutantpolypeptide, or fragment thereof. 2-3. (canceled)
 4. The method of claim1, wherein the TRPA1 function is an association with TRPV1.
 5. Themethod of claim 4, wherein the Tmem100 mutant polypeptide, or fragmentthereof, enhances the association of TRPA1 with TRPV1.
 6. The method ofclaim 1, wherein said TRPA1 function is an inward TRPA1-mediatedcurrent, an outward TRPA1-mediated current, TRPA1-mediated ion flux orTRPA1-mediated neuronal hyperexcitability.
 7. The method of claim 1,wherein the Tmem100 mutant polypeptide comprises a polypeptide with oneor more alterations in the amino acid sequence of SEQ ID NO: 1 or SEQ IDNO: 2, or fragments thereof.
 8. The method of claim 1, wherein theTmem100 mutant polypeptide comprises an amino acid sequence selectedfrom the group consisting of: SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7,SEQ ID NO: 8, and SEQ ID NO: 17, or fragments thereof.
 9. The method ofclaim 1, wherein the Tmem100 mutant polypeptide, or fragment thereof, isprovided to a cell and the cell is a sensory neuron.
 10. The method ofclaim 9, wherein the cell body of the sensory neuron resides in thedorsal root ganglia (DRG).
 11. The method of claim 1, used to prevent,treat, or alleviate symptoms of pain.
 12. The method of claim 1, used toprevent, treat, or alleviate symptoms of itch.
 13. The method of claim12, wherein the pain is acute pain or chronic pain.
 14. The method ofclaim 1, wherein the Tmem100 mutant polypeptide, or fragment thereof, isadministered in combination with one or more agents.
 15. The method ofclaim 1, wherein the Tmem100 mutant polypeptide, or fragment thereof, isadministered in combination with one or more of a TRPV1 inhibitor, aTRPV3 inhibitor, a TRPV4 inhibitor, or a TRPM8 inhibitor.
 16. Apharmaceutical composition for treating or preventing a conditioninvolving activation of TRPA1 or for which reduced TRPA1 activity canreduce the severity, comprising an effective amount of a Tmem100 mutantpolypeptide, or fragment thereof.
 17. The pharmaceutical composition ofclaim 16, wherein the Tmem100 mutant polypeptide comprises a polypeptidewith one or more alterations in the amino acid sequence of SEQ ID NO: 1or SEQ ID NO: 2, or fragments thereof.
 18. The pharmaceuticalcomposition of claim 16, wherein the Tmem100 mutant polypeptidecomprises an amino acid sequence selected from the group consisting of:SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8, or fragmentsthereof.
 19. An isolated polypeptide comprising an amino acid sequenceselected from the group consisting of: SEQ ID NO: 5, SEQ ID NO: 6, SEQID NO: 7 and SEQ ID NO: 8, or fragments thereof; or an isolatedpolypeptide encoded by a nucleic acid sequence comprising a nucleotidesequence selected from the group consisting of: SEQ ID NO: 9, SEQ ID NO:10, SEQ ID NO: 11 and SEQ ID NO: 12, or fragments thereof; or anisolated nucleic acid comprising a nucleotide sequence selected from thegroup consisting of: SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 and SEQID NO: 12, or fragments thereof; or an isolated nucleic acid comprisinga nucleotide sequence which encodes a polypeptide comprising an aminoacid sequence selected from the group consisting of: SEQ ID NO: 5, SEQID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8, or fragments thereof. 20-25.(canceled)